Methods and compositions for the treatment of motor neuron injury and neuropathy

ABSTRACT

Disclosed are therapeutic treatment methods, compositions and devices for maintaining neural pathways in a mammal, including enhancing survival of neurons at risk of dying, inducing cellular repair of damaged neurons and neural pathways, and stimulating neurons to maintain their differentiated phenotype. In one embodiment, the invention provides means for stimulating CAM expression in neurons. The invention also provides means for evaluating the status of nerve tissue, including means for detecting and monitoring neuropathies in a mammal. The methods, devices and compositions include a morphogen or morphogen-stimulating agent provided to the mammal in a therapeutically effective concentration.

RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. Ser. No.08/260,675, filed Jun. 16, 1994, which is a file wrapper continuation ofU.S. Ser. No. 08/126,100, filed Sep. 23, 1993, which is a file wrappercontinuation of U.S. Ser. No. 07/922,813, filed July 31, 1992 filed as acontinuation-in-part of U.S. Ser. No. 07/752,764 and copending U.S. Ser.No. 07/753,059, both filed Aug. 30, 1991 as continuations-in-part ofU.S. Ser. No. 07/667,274, filed March 11, 1991. The above-mentionedapplications are incorporated by reference herein.

BACKGROUND OF THE INVENTION

The mammalian nervous system comprises a peripheral nervous system (PNS)and a central nervous system (CNS, comprising the brain and spinalcord), and is composed of two principal classes of cells: neurons andglial cells. The glial cells fill the spaces between neurons, nourishingthem and modulating their function. Certain glial cells, such as Schwanncells in the PNS and oligodendrocytes in th e CNS, also provide a myelinsheath that surrounds neural processes. The myelin sheath enables rapidconduction along the neuron. In the peripheral nervous system, axons ofmultiple neurons may bundle together in order to form a nerve fiber.These, in turn, may be combined into fascicles or bundles.

During development, differentiating neurons from the central andperipheral nervous systems send out axons that grow and make contactwith specific target cells. In some cases, axons must cover enormousdistances; some grow into the periphery, whereas others are confinedwithin the central nervous system. In mammals, this stage ofneurogenesis is complete during the embryonic phase of life and neuronalcells do not multiply once they have fully differentiated.

A host of neuropathies have been identified that affect the nervoussystem. The neuropathies, which may affect neurons themselves orassociated glial cells, may result from cellular metabolic dysfunction,infection, exposure to toxic agents, autoimmunity, malnutrition, orischemia. In some cases, the cellular neuropathy is thought to inducecell death directly. In other cases, the neuropathy may inducesufficient tissue necrosis to stimulate the body's immune/inflammatorysystem and the immune response to the initial injury then destroysneural pathways.

Where the damaged neural pathway results from CNS axonal damage,autologous peripheral nerve grafts have been used to bridge lesions inthe central nervous system and to allow axons to make it back to theirnormal target area. In contrast to CNS neurons, neurons of theperipheral nervous system can extend new peripheral processes inresponse to axonal damage. This regenerative property of peripheralnervous system axons is thought to be sufficient to allow grafting ofthese segments to CNS axons. Successful grafting appears to be limited,however, by a number of factors, including the length of the CNS axonallesion to be bypassed, and the distance of the graft sites from the CNSneuronal cell bodies, with successful grafts occurring near the cellbody.

Within the peripheral nervous system, this cellular regenerativeproperty of neurons has limited ability to repair function to a damagedneural pathway. Specifically, the new axons extend randomly, and areoften misdirected, making contact with inappropriate targets that cancause abnormal function. For example, if a motor nerve is damaged,regrowing axons may contact the wrong muscles, resulting in paralysis.In addition, where severed nerve processes result in a gap of longerthan a few millimeters, e.g., greater than 10 millimeters (mm),appropriate nerve regeneration does not occur, either because theprocesses fail to grow the necessary distance, or because of misdirectedaxonal growth. Efforts to repair peripheral nerve damage by surgicalmeans has met with mixed results, particularly where damage extends overa significant distance. In some cases, the suturing steps used to obtainproper alignment of severed nerve ends stimulates the formulation ofscar tissue which is thought to inhibit axon regeneration. Even wherescar tissue formation has been reduced, as with the use of nerveguidance channels or other tubular prostheses, successful regenerationgenerally still is limited to nerve damage of less than 10 millimetersin distance. In addition, the reparative ability of peripheral neuronsis significantly inhibited where an injury or neuropathy affects thecell body itself or results in extensive degeneration of a distal axon.

Mammalian neural pathways also are at risk due to damage caused byneoplastic lesions. Neoplasias of both the neurons and glial cells havebeen identified. Transformed cells of neural origin generally lose theirability to behave as normal differentiated cells and can destroy neuralpathways by loss of function. In addition, the proliferating tumors mayinduce lesions by distorting normal nerve tissue structure, inhibitingpathways by compressing nerves, inhibiting cerbrospinal fluid or bloodsupply flow, and/or by stimulating the body's immune response.Metastatic tumors, which are a significant cause of neoplastic lesionsin the brain and spinal cord, also similarly may damage neural pathwaysand induce neuronal cell death.

One type of morphoregulatory molecule associated with neuronal cellgrowth, differentiation and development is the cell adhesion molecule(“CAM”), most notably the nerve cell adhesion molecule (N-CAM). The CAMsare members the immunoglobulin super-family. They mediate cell-cellinteractions in developing and adult tissues through homophilic binding,i.e., CAM-CAM binding on apposing cells. A number of different CAMs havebeen identified. Of these, the most thoroughly studied are N-CAM andL-CAM (liver cell adhesion molecules), both of which have beenidentified on all cells at early stages of development, as well as indifferent adult tissues. In neural tissue development, N-CAM expressionis believed to be important in tissue organization, neuronal migration,nerve-muscle tissue adhesion, retinal formation, synaptogenesis, andneural degeneration. Reduced N-CAM expression also is thought to beassociated with nerve dysfunction. For example, expression of at leastone form of N-CAM, N-CAM-180, is reduced in a mouse demyelinatingmutant. Bhat, Brain Res. 452: 373-377 (1988). Reduced levels of N-CAMalso have been associated with normal pressure hydrocephalus, Werdelin,Acta Neurol. Scand. 79: 177-181 (1989), and with type II schizophrenia.Lyons, et al., Biol. Psychiatry 23: 769-775 (1988). In addition,antibodies against N-CAM have been shown to disrupt functional recoveryin injured nerves. Remsen, Exp. Neurobiol. 110: 268-273 (1990).

Currently no satisfactory method exists to repair the damage caused bytraumatic injuries of motor neurons and diseases of motor neurons.

There are 15,000 to 18,000 new cases of spinal cord injury each year inthe United States. In addition, there are approximately 200,000survivors of spinal cord injury. The annual cost of care for thesepatients exceeds $7 billion. The pathophysiology following acute spinalcord trauma is a complex and not fully understood mechanism. The primarytissue damage caused by mechanical trauma occurs immediately and isirreversible. Allen, J. Am. Med. Assoc. 57: 878-880 (1911). Experimentalevidence indicates that much of the post-traumatic tissue damage is theresult of a reactive process that begins within minutes after the injuryand continues for days or weeks. Janssen, et al., Spine 14: 23-32 (1989)and Panter, et al., (1992). This progressive, self-destructive processincludes pathophysiological mechanisms such as hemorrhage,post-traumatic ischemia, edema, axonal and neuronal necrosis, anddemyelinization followed by cyst formation and infarction. For review,see Tator, et al., J. Neurosurg, 75: 15-26 (199 i) and Faden, Crit. Rev.Neurobiol. 7: 175-186 (1993). Proposed injurious factors includeelectrolyte changes whereby increased intracellular calcium initiates acascade of events (Young, J. Neurotrauma 9, Suppl. 1: S9-S25 (1992) andYoung, J. Emerg. Med. 11: 13-22 (1993)), biochemical changes withuncontrolled transmitter release (Liu, et al., Cell 66: 807-815 (1991)and Yanase, et al., J. Neurosurg. 83: 884-888 (1995), arachidonic acidrelease, free-radical production, lipid peroxidation (Braughler, et al,J. Neurotrauma 9, Suppl. 1: S1-S7 (1992), eicosanoid production(Demediuk, et al., J. Neurosci. Res. 20: 115-121 (1988), endogenousopioids (Faden, et al., Ann Neurol. 17: 386-390 (1985), metabolicchanges including alterations in oxygen and glucose (Faden, Crit. Rev.Neurobiol. 7: 175-186 (1993)), inflammatory-changes (Blight, J.Neurotrauma 9, Suppl. 1: S83-S91 (1992), and astrocytic edema(Kinelberg, J. Neurotrauma 9, Suppl. 1: S71-S81 (1992). For the past 400years surgical approaches including laminectomy and decompression,accompanied by fusion, have been the most commonly practiced treatmentstrategies. Hansebout, “Early Management of Acute Spinal Cord Injury”,pp. 181-196 (1982) and Janssen, et al., Spine 14: 23-32 (1989). However,these procedures have not involved the application of techniques toaugment the regenerative properties of spinal cord tissue.

A host of diseases of motor neurons have been identified, includingdemyelinating diseases, myelopathies, and diseases of motor neurons suchas amyotrophic lateral sclerosis (ALS). INTERNAL MEDICINE, ch. 121-123(4th ed., J. H. Stein, ed., Mosby, 1994). Multiple sclerosis (MS) is themost common demyelinating disorder of the central nervous system,causing patches of sclerosis (i.e., plaques) in the brain and spinalcord. MS has protean clinical manifestations, depending upon thelocation and size of the plaque. Typical symptoms include visual loss,diplopia, nystagmus, dysarthria, weakness, paresthesias, bladderabnormalities, and mood alterations. Myriad treatments have beenproposed for this long-term variable illness. The list of proposedtreatments encompasses everything from diet to electrical stimulation toacupuncture, emotional support, and various forms of immunosupressivetherapy. None have proved to be satisfactory.

Progressive loss of lower and upper motor neurons occurs in severaldiseases (e.g., primary lateral sclerosis, spinal muscular atrophy,benign focal amyotrophy). However, ALS is the most common form of motorneuron disease. Loss of both lower and upper motor neurons occur in ALS.Symptoms include progressive skeletal muscle wasting, weakness,gasciculations, and cramping. Some cases have predominant involvement ofbrainstem motoneurons (progressive bulbar palsy). Unfortunately,treatment of motor neuron and related diseas is largely supportive atthis time. INTERNAL MEDICINE, ch. 123 (4th ed., J. H. Stein, ed., Mosby,1994).

Accordingly, there is a need in the art for treatments of motor neuronsdisorders and injuries, and related deficits in neural functions.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for maintainingneural pathways in a mammal in vivo, including methods for enhancing thesurvival of neural cells.

In a preferred embodiment, methods of the invention for treating motorneuron defects, including amyotrophic lateral sclerosis, multiplesclerosis, and spinal cord injury comprise administering a morphogencomprising a dimeric protein having an amino acid sequence selected fromthe group consisting of a sequence have 70% homology with the C-terminalseven-cysteine skeleton of human OP-1 (amino acids 330-341 of SEQ ID NO:2), a sequence having greater than 60% amino acid sequence identity withhuman OP-1; generic sequence 7 (SEQ ID NO: 4); generic sequence 8 (SEQID NO: 6); generic sequence 10 (SEQ ID NO: 7); and OPX (SEQ ID NO: 3);wherein the morphogen stimulates production of N-CAM or L1 isoforms byan NG108-15 cell in vivo. Spinal cord injuries include injuriesresulting from a tumor, mechanical trauma, and chemical trauma. The sameor similar methods are contemplated to restore motor function in amammal having amyotrophic lateral sclerosis, multiple sclerosis, or aspinal cord injury. Administering one of the aforementioned morphogensalso provides a prophylactic function. Such administration has theeffect of preserving motor function in a mammal having, or at risk ofhaving, amyotrophic lateral sclerosis, multiple sclerosis, or a spinalcord injury. Also according to the invention, morphogen administrationpreserves the integrity of the nigrostriatal pathway.

Specifically, methods of the invention for treating (pre- orpost-symptomatically) amyotrophic lateral sclerosis, multiple sclerosis,or a spinal cord injury comprise administering a morphogen selected fromthe group consisting of human OP-1, mouse OP-1, human OP-2, mouse OP-2,60A, GDF-1, BMP2A, BMP2B, DPP, Vgl, Vgr-1, BMP3, BMP5, and BMP6. Suchmorphogens are capable of stimulating production of N-CAM or L1 isoformby an NG108-15 cell in vivo.

In a particularly-preferred embodiment, the morphogen is a solublecomplex, comprising at least one morphogen pro domain, or fragmentthereof, non-covalently attached to a mature morphogen.

In one aspect, the invention features compositions and therapeutictreatment methods comprising administering to a mammal a therapeuticallyeffective amount of a morphogenic protein (“morphogen”), as definedherein, upon injury to a neural pathway, or in anticipation of suchinjury, for a time and at a concentration sufficient to maintain theneural pathway, including repairing damaged pathways, or inhibitingadditional damage thereto.

In another aspect, the invention features compositions and therapeutictreatment methods for maintaining neural pathways. Such treatmentmethods include administering to the mammal, upon injury to a neuralpathway or in anticipation of such injury, a compound that stimulates atherapeutically effective concentration of an endogenous morphogen.These compounds are referred to herein as morphogen-stimulating agents,and are understood to include substances which, when administered to amammal, act on tissue(s) or organ(s) that normally are responsible for,or capable of, producing a morphogen and/or secreting a morphogen, andwhich cause endogenous level of the morphogen to be altered.

In particular, the invention provides methods for protecting neuronsfrom the tissue destructive effects associated with the body's immuneand inflammatory response to nerve injury. The invention also providesmethods for stimulating neurons to maintain their differentiatedphenotype, including inducing the redifferentiation of transformed cellsof neuronal origin to a morphology characteristic of untransformedneurons. In one embodiment, the invention provides means for stimulatingproduction of cell adhesion molecules, particularly nerve cell adhesionmolecules (N-CAM). The invention also provides methods, compositions anddevices for stimulating cellular repair of damaged neurons and neuralpathways, including regenerating damaged dendrites or axons. Inaddition, the invention also provides means for evaluating the status ofnerve tissue, and for detecting and monitoring neuropathies bymonitoring fluctuations in morphogen levels.

In one aspect of the invention, the morphogens described herein areuseful in repairing damaged neural pathways of the peripheral nervoussystem. In particular, morphogens are useful for repairing damagedneural pathways, including transected or otherwise damaged nerve fibers.Specifically, the morphogens described herein are capable of stimulatingcomplete axonal nerve regeneration, including vascularization andreformation of the myelin sheath. Preferably, the morphogen preferablyis provided to the site of injury in a biocompatible, bioresorbablecarrier capable of maintaining the morphogen at the site and, wherenecessary, means for directing axonal growth from the proximal to thedistal ends of a severed neuron. For example, means for directing axonalgrowth may be required where nerve regeneration is to be induced over anextended distance, such as greater than 10 mm. Many carriers capable ofproviding these functions are envisioned. For example, useful carriersinclude substantially insoluble materials or viscous solutions preparedas disclosed herein comprising laminin, hyaluronic acid or collagen, orother suitable synthetic, biocompatible polymeric materials such aspolylactic, polyglycolic or polybutyric acids and/or copolymers thereof.A preferred carrier comprises an extracellular matrix compositionderived, for example, from mouse sarcoma cells.

In a particularly preferred embodiment, a morphogen is disposed in anerve guidance channel which spans the distance of the damaged pathway.The channel acts both as a protective covering and a physical means forguiding growth of a neurite. Useful channels comprise a biocompatiblemembrane, which may be tubular in structure, having a dimensionsufficient to span the gap in the nerve to be repaired, and havingopenings adapted to receive severed nerve ends. The membrane may be madeof any biocompatible, nonirritating material, such as silicone or abiocompatible polymer, such as polyethylene or polyethylene vinylacetate. The casing also may be composed of biocompatible, bioresorbablepolymers, including, for example, collagen, hyaluronic acid, polylactic,polybutyric, and polyglycolic acids. In a preferred embodiment, theouter surface of the channel is substantially impermeable.

The morphogen may be disposed in the channel in association with abiocompatible carrier material, or it may be adsorbed to or otherwiseassociated with the inner surface of the casing, such as is described inU.S. Pat. No. 5,011,486, provided that the morphogen is accessible tothe severed nerve ends.

Morphogens described herein are also useful in autologous peripheralnerve segment implants, such as in the repair of damaged or detachedretinas, or other damage to the optic nerve.

In another aspect of the invention, morphogens described herein areuseful to protect against damage associated with the body'simmune/inflammatory response to an initial injury to nerve tissue. Sucha response may follow trauma to nerve tissue, caused, for example, by anautoimmune dysfunction, neoplastic lesion, infection, chemical ormechanical trauma, disease, by interruption of blood flow to the neuronsor glial cells, or by other trauma to the nerve or surrounding material.For example, the primary damage resulting from hypoxia orischemia-reperfusion following occlusion of a neural blood supply, as inan embolic stroke, is believed to be immunologically associated. Inaddition, at least part of the damage associated with a number ofprimary brain tumors also appears to be immunologically related.Application of a morphogen, either directly or systemically alleviateand/or inhibit the immunologically related response to a neural injury.Alternatively, administration of an agent capable of stimulatingmorphogen expression and/or secretion in vivo, preferably at the site ofinjury, may also be used. Where the injury is to be induced, as duringsurgery or other aggressive clinical treatment, the morphogen or agentmay be provided prior to induction of the injury to provide aneuroprotective effect to the nerve tissue at risk.

Generally, morphogens useful in methods and compositions of theinvention are dimeric proteins that induce morphogenesis of one or moreeukaryotic (e.g., mammalian) cells, tissues or organs. Tissuemorphogenesis includes de novo or regenerative tissue formation, such asoccurs in a vertebrate embryo during development. Of particular interestare morphogens that induce tissue-specific morphogenesis at least ofbone or neural tissue. As defined herein, a morphogen comprises a pairof polypeptides that, when folded, form a dimeric protein that elicitsmorphogenetic responses in cells and tissues displayingmorphogen-specific receptors. That is, the morphogens generally induce acascade of events including all of the following in a morphogenicallypermissive environment: stimulating proliferation of progenitor cells;stimulating the differentiation of progenitor cells; stimulating theproliferation of differentiated cells; and, supporting the growth andmaintenance of differentiated cells. “Progenitor” cells are uncommittedcells that are competent to differentiate into one or more specifictypes of differentiated cells, depend ing on their genomic repertoireand the tissue specificity of the permissive environment in whichmorphogenesis is induced. An exemplary progenitor cell is ahematopoeitic stem cell, a mesenchymal stem cell, a basement epitheliumcell, a neural crest cell, or the like. Further, morphogens can delay ormitigate the onset of senescence- or quiescence-associated loss ofphenotype and/or tissue function. Still further, morphogens canstimulate phenotypic expression of a differentiated cell type, includingexpression of metabolic and/or functional, e.g., secretory, propertiesthereof. In addition, morphogens can induce redifferentiation ofcommitted cells (e.g., osteoblasts, neuroblasts, or the like) underappropriate conditions. As noted above, morphogens that induceproliferation and/or differentiation at least of bone or neural tissue,and/or support the growth, maintenance and/or functional properties ofneural tissue, are of particular interest herein. See, e.g., WO92/15323, WO 93/04692, WO 94/03200 (providing more detailed disclosuresas to the tissue morphogenic properties of these proteins).

As used herein, the terms “morphogen,” “bone morphogen,” “bonemorphogenic protein,” “BMP,” “morphogenic protein” and “morphogeneticprotein” all embrace the class of proteins typified by human osteogenicprotein 1 (hOP-1). Nucleotide and amino acid sequences for hOP-1 areprovided in SEQ ID NOS: 1 and 2, respectively. For ease of description,hOP—I is considered a representative morphogen. It will be appreciatedthat OP-1 is merely representative of the TGF-β subclass of true tissuemorphogens and is not intended to limit the description. Other known anduseful morphogens include, but are not limited to, BMP-2, BMP-3, BMP-3b,BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13,BMP-15, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10,GDF-11, GDF-12, 60A, NODAL, UNIVIN, SCREW, ADMP, and NEURAL, andmorphogenically-active amino acid variants of any thereof.

In specific embodiments, useful morphogens include those sharing theconserved seven cysteine skeleton, and sharing at least 70% amino acidsequence homology (similarity), within the C-terminal seven-cysteineskeleton of human OP-1, residues 330-431 of SEQ ID NO: 2 (hereinafterreferred to as the “reference sequence”). In another embodiment, theinvention encompasses use of biologically active species (phylogenetic)variants of any of the morphogenic proteins recited herein, includingconservative amino acid sequence variants, proteins encoded bydegenerate nucleotide sequence variants, and morphogenically-activeproteins sharing the conserved seven cysteine skeleton as defined hereinand encoded by a DNA competent to hybridize under standard stringencyconditions to a DNA encoding a morphogenic protein disclosed herein,including, without limitation, OP-1 or BMP-2 or BMP-4. Presently,however, the reference sequence is that of residues 330-431 of SEQ IDNO: 2 (OP-1).

In still another embodiment, morphogens useful in methods andcompositions of the invention are defined as morphogenically-activeproteins having any one of the generic sequences defined herein,including OPX (SEQ ID NO: 3) and Generic Sequences 7 and 8 (SEQ ID NOS:4 and 5, respectively), or Generic Sequences 9 and 10 (SEQ ID NOS: 6 and7, respectively). OPX encompasses the observed variation between theknown phylogenetic counterparts of the osteogenic OP-1 and OP-2proteins, and is described by the amino acid sequence presented hereinbelow and in SEQ ID NO: 3. Generic Sequence 9 is a 96 amino acidsequence containing the C-terminal six cysteine skeleton observed inhOP-1 (residues 335-431 of SEQ ID NO: 2) and wherein the remainingresidues encompass the observed variation among OP-1, OP-2, OP-3, BMP-2,BMP-3, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-11, BMP-15, GDF-1,GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, 60A, UNIVIN,NODAL, DORSALIN, NEURAL, SCREW and ADMP. That is, each of thenon-cysteine residues is independently selected from the correspondingresidue in this recited group of known, naturally-sourced proteins.Generic Sequence 10 is a 102 amino acid sequence which includes a fiveamino acid sequence added to the N-terminus of the Generic Sequence 9and defines the seven cysteine skeleton observed in hOP-1 (330-431 SEQID NO: 2). Generic Sequences 7 and 8 are 96 and 102 amino acidsequences, respectively, containing either the six cysteine skeleton(Generic Sequence 7) or the seven cysteine skeleton (Generic Sequence 8)defined by hOP-1 and wherein the remaining non-cysteine residuesencompass the observed variation among OP-1, OP-2, OP-3, BMP-2, BMP-3,BMP4, 60A, DPP, Vgl, BMP-5, BMP-6, Vgr-1, and GDF-1.

Of particular interest are morphogens which, when provided to a specifictissue of a mammal, induce tissue-specific morphogenesis or maintain thenormal state of differentiation and growth of that tissue. In preferredembodiments, the present morphogens induce the formation of vertebrate(e.g., avian or mammalian) body tissues, such as but not limited tonerve, eye, bone, cartilage, bone marrow, ligament, tooth dentin,periodontium, liver, kidney, lung, heart, or gastrointestinal lining.Preferred methods may be carried out in the context of developingembryonic tissue, or at an aseptic, unscarred wound site inpost-embryonic tissue. Methods of identifying such morphogens, ormorphogen receptor agonists, are known in the art and include assays forcompounds which induce morphogen-mediated responses (e.g., induction ofendochondral bone formation, induction of differentiation of metanephricmesenchyme, and the like). In a preferred embodiment, morphogens of theinvention, when implanted in a mammal in conjunction with a matrixpermissive of bone morphogenesis, are capable of inducing adevelopmental cascade of cellular and molecular events that culminatesin endochondral bone formation. See, U.S. Pat. No. 4,968,590; Sampath,et al., Proc. Natl. Acad. Sci. USA 80: 6591-6595 (1983), the disclosuresof which are incorporated by reference herein.

In an alternative preferred embodiment, morphogens of the invention arealso capable of stimulating production of cell adhesion molecules,including nerve cell adhesion molecules (N-CAMs). In a preferredembodiment, the present morphogens are capable of stimulating theproduction of N-CAM in vitro in NG108-15 cells, which are a preferredmodel system for assessing neuronal differentiation, particularly motorneuron differentiation.

In still other embodiments, an agent which acts as an agonist of amorphogen receptor may be administered instead of the morphogen itself.An “agonist” of a receptor is a compound which binds to the receptor,and for which the result of such binding is similar to the result ofbinding the natural, endogenous ligand of the receptor. That is, thecompound must, upon interaction with the receptor, produce the same orsubstantially similar transmembrane and/or intracellular effects as theendogenous ligand. Thus, an agonist of a morphogen receptor binds to thereceptor and such binding has the same or a functionally similar resultas morphogen binding (e.g., induction of morphogenesis). The activity orpotency of an agonist can be less than that of the natural ligand, inwhich case the agonist is said to be a “partial agonist,” or it can beequal to or greater than that of the natural ligand, in which case it issaid to be a “full agonist.” Thus, for example, a small peptide or othermolecule which can mimic the activity of a morphogen in binding to andactivating the morphogen's receptor may be employed as an equivalent ofthe morphogen. Preferably the agonist is a full agonist, but partialmorphogen receptor agonists may also be advantageously employed. Such anagonist may also be referred to as a morphogen “mimic,” “mimetic,” or“analog.”

Morphogen inducers and agonists can be identified by mutation,site-specific mutagenesis, combinatorial chemistry, etc. Such methodsare well known in the art. For example, methods of identifying morphogeninducers or agonists of morphogen receptors may be found in U.S. Ser.No. 08/478,097 filed Jun. 7, 1995 and U.S. Ser. No. 08/507,598 filedJul. 26, 1995, the disclosures of which are incorporated herein byreference. Candidate morphogen inducers and agonists are then tested fortheir ability to induce endochondral bone formation and preferably, tostimulate N-CAM production in neurons or in a neuronal model system,such as NG108-15 cells. Morphogen inducers and agonists identifiedaccording to the present invention are capable of inducing endochondralbone formation when implanted in a mammal in conjunction with a matrixpermissive of bone morphogenesis and are capable of stimulatingproduction of N-CAM in vitro.

The preferred methods, material, and examples that will now be describedare illustrative only and are not intended to be limiting. Otherfeatures and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a tabular presentation of the percent amino acid sequenceidentity and percent amino acid sequence homology (“similarity”) thatvarious members of the family of morphogenic proteins as defined hereinshare with hOP-1 in the C-terminal seven cysteine skeleton;

FIG. 2 (Panels A and B) are photographs illustrating the ability ofmorphogen (OP-1) to induce transformed neuroblastoma×glioma cells (Panel1A) to redifferentiate to a morphology characteristic of untransformedneurons (Panel 1B);

FIG. 3A is a line graph depicting a dose response curve for theinduction of the 180 kDa and 140 kDa N-CAM isoforms in morphogen-treatedNG108-15 cells;

FIG. 3B is a photograph of a Western blot of whole cell extracts frommorphogen-treated NG108-15 cells with an N-CAM-specific antibody; and

FIG. 4 is a line graph depicting the mean number of cell aggregatescounted in twenty (20) randomly selected magnified viewing fields as afunction of morphogen concentration.

FIG. 5 is a photograph of an immunoblot demonstrating the presence ofOP-1 in human serum.

FIG. 6 is a bar graph comparing the effects of OP-1 and glial cells onaxonal and dendritic length after one day in vitro.

FIG. 7 is a bar graph comparing the effects of OP-1 and glial cells onaxonal and dendritic length after three days in vitro.

FIG: 8 is a bar graph comparing the effects of OP-1 and glial cells ondendritic branching after three days in vitro.

FIG. 9 (Panels A-C) are line graphs depicting a time course of theresponse of cultured sympathetic neurons to OP-1. Intracellular dyeinjections (N>30 for each point) were performed at various times todetermine: the percentage of cells with dendrites (Panel A); the meannumber of dendrites/cell (Panel B); and the number of axons/cell (PanelC). The bars shown in Panel B represent the SEM; where bars are notshown, the SEM was smaller than the size of the symbol. Open symbols,control; filled symbols, cells supplemented with 100 ng/ml OP-1 duringthe time course study.

FIG. 10 is a line graph depicting the effects of varying concentrationsof OP-1 on dendritic growth. Sympathetic neurons were exposed to OP-1 inculture for three days and then immunostained with a dendrite-specificmAb (SMI 32). Percentage of cells with dendrites, open circles; meannumber of dendrites per cell, filled circles.

FIG. 11 is a line graph depicting the effects of varying concentrationsof different morphogens on dendritic growth. Sympathetic neurons wereexposed to various concentrations of BMP-2, OP-1, 60A, BMP-3 or CDMP-2beginning on the fifth day in vitro and then immunostained on day 10with a dendrite-specific antibody (SMI 32). Data are presented as themean±SEM. N=30.

FIG. 12 is a bar graph comparing the effects of OP-1 and glial cells onsynapse formation after three and four days in vitro.

FIG. 13 is a bar graph depicting the effects of OP-1 treatment on thesize of spinal cord neurons transplanted intra-ocularly in vivo over aperiod of four weeks.

FIG. 14 is a photograph of the neurofilament staining of intra-ocularcultures. Spinal cord transplant cultures were stained four weekspost-grafting. Cultures were treated with weekly injections of vehicleor OP-1.

FIG. 15 is a photograph of the choline acetyltransferase staining ofintra-ocular cultures. Spinal cord transplant cultures were stained fourweeks post-grafting. Cultures were treated with weekly injections ofvehicle or OP-1.

FIG. 16 is a bar graph depicting injury severity scores in the forelimbplacing task of sham animals (N=7; black bars), vehicle-treated animalswith traumatic brain injury (N=8; grey bars), and OP-1-treated animalswith traumatic brain injury (10 μg/intracistemal injection; total OP-1delivered in 2 injections=20 μg/animal; N=7; white bars).

FIG. 17 is a bar graph depicting failure scores in the beam walk task ofsham animals (N=5; black bars), vehicle-treated animals with traumaticbrain injury (N=5; grey bars), and OP-1-treated animals with traumaticbrain injury (10 μg/intracisternal injection; total OP-1 delivered in 2injections=20 μg/animal; N=4; white bars).

FIG. 18 is a bar graph depicting beam latency scores of sham animals(N=5; black bars), vehicle-treated animals with traumatic brain injury(N=5; grey bars), and OP-1-treated animals with traumatic brain injury(10 μg/intracistemal injection; total OP-1 delivered in 2 injections=20μg/animal; N=4; white bars).

DETAILED DESCRIPTION OF THE INVENTION

It has now been discovered that morphogens enhance survival of neurons,and maintain neural pathways. As described herein, morphogens arecapable of enhancing survival of neurons, stimulating neuronal CAMexpression, maintaining the phenotypic expression of differentiatedneurons, inducing the redifferentiation of transformed cells of neuralorigin, and stimulating axonal growth over breaks in neural processes,particularly large gaps in axons. Morphogens also protect against tissuedestruction associated with immunologically-related nerve tissue damage.Finally, morphogens may be used as part of a method for monitoring theviability of nerve tissue in a mammal.

A. Biochemical, Structural and Functional Properties of UsefulMorphogenic Proteins

As noted above, a protein is morphogenic as defined herein if it inducesthe developmental cascade of cellular and molecular events thatculminate in the formation of new, organ-specific tissue. In a preferredembodiment, a morphogen is a dimeric protein, each polypeptide componentof which has a sequence that corresponds to, or is functionallyequivalent to, at least the conserved C-terminal six or seven cysteineskeleton of human OP-1, included in SEQ ID NO: 2, and/or which shares70% amino acid sequence homology with OP-1 in this region. Themorphogens are generally competent to induce a cascade of eventsincluding the following, in a morphogenically permissive environment:stimulating proliferation of progenitor cells; stimulating thedifferentiation of progenitor cells; stimulating the proliferation ofdifferentiated cells; and supporting the growth and maintenance ofdifferentiated cells. Under appropriate conditions morphogens are alsocompetent to induce redifferentiation of cells that have undergoneabnormal differentiation. Details of how the morphogens useful in thisinvention were identified, as well as a description on how to make, useand test them for morphogenic activity are disclosed in numerouspublications, including U.S. Pat. Nos. 5,011,691 and 5,266,683, and theinternational patent application publications WO 92/15323; WO 93/04692;and WO 94/03200, each of which are incorporated by reference herein. Asdisclosed therein, the morphogens can be purified from naturally-sourcedmaterial or recombinantly produced from prokaryotic or eukaryotic hostcells, using the genetic sequences disclosed therein. Alternatively,novel morphogenic sequences can be identified following the proceduresdisclosed therein.

The naturally-occurring morphogens share substantial amino acid sequencehomology in their C-terminal sequences (sharing e.g., a six or sevencysteine skeleton sequence). Typically, a naturally-occurring morphogenis translated as a precursor, having an N-terminal signal peptidesequence, typically less than about 35 residues in length, followed by a“pro” domain that is cleaved to yield the mature polypeptide, whichincludes the biologically active C-terminal skeleton sequence. Thesignal peptide is cleaved rapidly upon translation, at a cleavage sitethat can be predicted in a given sequence using the method of VonHeijne, Nucleic Acids Research 14: 4683-4691 (1986). The pro polypeptidetypically is about three times larger than the fully processed, matureC-terminal polypeptide. Under native conditions, the protein is secretedas a mature dimer and the cleaved pro polypeptide is thought to remainassociated therewith to form a protein complex, presumably to improvethe solubility of the mature dimeric protein. The complexed form of amorphogen is generally observed to be more soluble than the mature formunder physiological conditions.

Natural-sourced morphogenic protein in its mature, native form, istypically a glycosylated dimer, having an apparent molecular weight ofabout 30-36 kDa as determined by SDS-PAGE. When reduced, the 30 kDaprotein gives rise to two glycosylated polypeptide subunits havingapparent molecular weights in the range of about 16 kDa and about 18kDa. The unglycosylated dimeric protein, which also has morphogenicactivity, typically has an apparent molecular weight in the range ofabout 27 kDa. When reduced, the 27 kDa protein gives rise to twounglycosylated polypeptides having molecular weights typically in therange of about 14 kDa to about 16 kDa.

In preferred embodiments, each of the polypeptide subunits of a dimericmorphogenic protein as defined herein comprises an amino acid sequencesharing a defined relationship with an amino acid sequence of areference morphogen. In one embodiment, preferred morphogenicpolypeptide chains share a defined relationship with a sequence presentin morphogenically-active human OP-1, SEQ ID NO: 2. However, any one ormore of the naturally-occurring or biosynthetic morphogenic proteinsdisclosed herein similarly could be used as a reference sequence.Preferred morphogenic polypeptide chains share a defined relationshipwith at least the C-terminal six cysteine skeleton of human OP-1,residues 335-431 of SEQ ID NO: 2. Preferably, morphogenic proteins sharea defined relationship with at least the C-terminal seven cysteineskeleton of human OP-1, residues 330-431 of SEQ ID NO: 2.

Functionally equivalent sequences include functionally equivalentarrangements of cysteine residues disposed within the referencesequence, including amino acid insertions or deletions which alter thelinear arrangement of these cysteines, but do not materially impairtheir relationship in the folded structure of the dimeric morphogenprotein, including their ability to form such intra- or inter-chaindisulfide bonds as may be necessary for morphogenic activity. Forexample naturally-occurring morphogens have been described in which atleast one internal deletion (of one residue; BMP2) or insertion (of fourresidues; GDF-1) is present but does not abrogate biological activity.Functionally equivalent sequences further include those wherein one ormore amino acid residues differ from the corresponding residue of areference sequence, e.g., the C-terminal seven cysteine skeleton ofhuman OP-1, provided that this difference does not destroy tissuemorphogenic activity. Accordingly, conservative substitutions ofcorresponding amino acids in the reference sequence are preferred. Aminoacid residues that are “conservative substitutions” for correspondingresidues in a reference sequence are those that are physically orfunctionally similar to the corresponding reference residues, e.g., thathave similar size, shape, electric charge, chemical properties includingthe ability to form covalent or hydrogen bonds, or the like.Particularly preferred conservative substitutions are those fulfillingthe criteria defined for an accepted point mutation in Dayhoff, et al.,5 ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, Suppl. 3, ch. 22 pp. 354-352(1978), Natl. Biomed. Res. Found., Washington, D.C. 20007, the teachingsof which are incorporated by reference herein. Examples of conservativesubstitutions include the substitution of one amino acid for anotherwith similar characteristics, e.g., substitutions within the followinggroups: valine, glycine; glycine, alanine; valine, isoleucine, leucine;aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine. The term “conservativesubstitution” also includes the use of a synthetic or derivatized aminoacid in place of the corresponding natural parent amino acid, providedthat antibodies raised to the resulting variant polypeptide alsoimmunoreact with the corresponding naturally sourced morphogenpolypeptide.

The following publications disclose publications morphogen polypeptidesequences, as well as relevant chemical and physical properties, ofnaturally-occurring and/or synthetic morphogens: OP-1 and OP-2: U.S.Pat. No. 5,011,691, U.S. Pat. No. 5,266,683, Ozkaynak, et al., EMBO J.9: 2085-2093 (1990); OP-3: WO 94/10203 (PCT US93/10520); BMP-2, BMP-3,and BMP-4: WO 88/00205, Wozney, et al., Science 242: 1528-1534 (1988);BMP-5 and BMP-6: Celeste, et al., PNAS 87: 9843-9847 (1991); Vgr-1:Lyons, et al, PNAS 86: 4554-4558 (1989); DPP: Padgett, et al., Nature325: 81-84 (1987); Vg-1: Weeks Cell 51: 861-867 (1987); BMP-9: WO95/33830 (PCT/US95/07084); BMP-10: WO 94/26893 (PCT/US94/05290); BMP-11:WO 94/26892 (PCT/US94/05288); BMP-12: WO 95/16035 (PCT/US94/14030);BMP-13: WO 95/16035 (PCT/US94/14030); GDF-1: WO 92/00382(PCT/US91/04096) and Lee, et al., PNAS 88: 42504254 (1991); GDF-8: WO94/21681 (PCT/US94/03019); GDF-9: WO 94/15966 (PCT/US94/00685); GDF-10:WO 95/10539 (PCT/US94/11440); GDF-11: WO 96/01845 (PCT/US95/08543);BMP-15: WO 96/36710 (PCT/US96/06540); MP121: WO 96/01316(PCT/EP95/02552); GDF-5 (CDMP-1, MP52): WO 94/15949 (PCT/US94/00657) andWO 96/14335 (PCT/US94/12814) and WO 93/16099 (PCT/EP93/00350); GDF-6(CDMP-2, BMP-13): WO 95/01801 (PCT/US94/07762) and WO 96/14335 and WO95/10635 (PCT/US94/14030); GDF-7 (CDMP-3, BMP-12): WO 95/10802(PCT/US94/07799) and WO 95/10635 (PCT/US94/14030). In anotherembodiment, useful proteins include biologically active biosyntheticconstructs, including novel biosynthetic morphogenic proteins andchimeric proteins designed using sequences from two or more knownmorphogens. See also the biosynthetic constructs disclosed in U.S. Pat.No. 5,011,691, the disclosure of which is incorporated herein byreference (e.g., COP-1, COP-3, COP-4, COP-5, COP-7, and COP-16).

In certain preferred embodiments, useful morphogenic proteins includethose in which the amino acid sequences comprise a sequence sharing atleast 70% amino acid sequence homology or “similarity”, and preferably80% homology or similarity, with a reference morphogenic proteinselected from the exemplary, naturally-occurring morphogenic proteinslisted herein. Preferably, the reference protein is human OP-1, and thereference sequence thereof is the C-terminal seven cysteine skeletonpresent in osteogenically active forms of human OP-1, residues 330-431of SEQ ID NO: 2. Useful morphogenic proteins accordingly includeallelic, phylogenetic counterpart and other variants of the preferredreference sequence, whether naturally-occurring or biosyntheticallyproduced (e.g., including “muteins” or “mutant proteins”), as well asnovel members of the general morphogenic family of proteins includingthose set forth and identified above. Certain particularly preferredmorphogenic polypeptides share at least 60% amino acid identity with thepreferred reference sequence of human OP-1, still more preferably atleast 65% amino acid identity therewith.

In certain embodiments, a poly eptide suspected of being functionallyequivalent to a reference morphogen polypeptide is aligned therewithusing the method of Needleman, et al., J. Mol. Biol. 48: 443-453 (1970),implemented conveniently by computer programs such as the Align program(DNAstar, Inc.). As noted above, internal gaps and amino acid insertionsin the candidate sequence are ignored for purposes of calculating thedefined relationship, conventionally expressed as a level of amino acidsequence homology, or identity, between the candidate and referencesequences. “Amino acid sequence homology” is understood herein toinclude both amino acid sequence identity and similarity. Homologoussequences share identical and/or similar amino acid residues, wheresimilar residues are conservation substitutions for, or “allowed pointmutations” of, corresponding amino acid residues in an aligned referencesequence. Thus, a candidate polypeptide sequence that shares 70% aminoacid homology with a reference sequence is one in which any 70% of thealigned residues are either identical to, or are conservativesubstitutions of, the corresponding residues in a reference sequence. Ina preferred embodiment, the reference sequence is the C-terminal sevencysteine skeleton sequence of human OP-1.

FIG. 1 recites the percent amino acid sequence homology (similarity) andpercent identity within the C-terminal seven cysteine skeletons ofvarious representative members of the TGF-β family, using OP-1 as thereference sequence. The percent homologies recited in the figure aredetermined by aligning the sequences essentially following the method ofNeedleman, et al., J. Mol. Biol., 48: 443-453 (1970), and using theAlign Program (DNAstar, Inc.). Insertions and deletions from thereference morphogen sequence (the C-terminal, biologically activeseven-cysteine skeleton of hOP-1) are ignored for purposes ofcalculation.

As is apparent to one of ordinary skill in the art reviewing thesequences for the proteins listed in FIG. 1, significant amino acidchanges can be made from the reference sequence while retainingsubstantial morphogenic activity. For example, while the GDF-1 proteinsequence shares only about 50% amino acid identity with the hOP-1sequence described herein, the GDF-1 sequence shares greater than 70%amino acid sequence homology with the hOP-1 sequence, where “homology”is as defined above. Moreover, GDF-1 contains a four amino acid insert(Gly-Gly-Pro-Pro) between the two residues corresponding to residue 372and 373 of OP-1 (SEQ ID NO: 2). Similarly, BMP-3 has a “extra” residue,a valine, inserted between the two residues corresponding to residues385 and 386 of hOP-1 (SEQ ID NO: 2). Also, BMP-2 and BMP-4 are both“missing” the amino acid residue corresponding to residue 389 of OP-1(SEQ ID NO: 2). None of these “deviations” from the reference sequenceappear to interfere substantially with biological activity.

In other preferred embodiments, the family of morphogenic polypeptidesuseful in the present invention, and members thereof, are defined by ageneric amino acid sequence. For example, Generic Sequence 7 (SEQ ID NO:4) and Generic Sequence 8 (SEQ ID NO: 5) disclosed below, encompass theobserved variations between preferred protein family members identifiedto date, including at least OP-1, OP-2, OP-3, CBMP-2A, CBMP-2B, BMP-3,60A, DPP, Vg1, BMP-5, BMP-6, Vgr-1, and GDF-1. The amino acid sequencesfor these proteins are described herein and/or in the art, as summarizedabove. The generic sequences include both the amino acid identity sharedby these sequences in the C-terminal skeleton, defined by the six andseven cysteine skeletons (Generic Sequences 7 and 8, respectively), aswell as alternative residues for the variable positions within thesequence. The generic sequences provide an appropriate cysteine skeletonwhere inter- or intramolecular disulfide bonds can form, and containcertain critical amino acids likely to influence the tertiary structureof the folded proteins. In addition, the generic sequences allow for anadditional cysteine at position 36 (Generic Sequence 7) or position 41(Generic Sequence 8), thereby encompassing the morphogenically-activesequences of OP-2 and OP-3. Generic Sequence 7 (SEQ ID NO: 4) Leu XaaXaa Xaa Phe Xaa Xaa 1 5 Xaa Gly Trp Xaa Xaa Xaa Xaa Xaa Xaa Pro 10 15Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly 20 25 Xaa Cys Xaa Xaa Pro XaaXaa Xaa Xaa Xaa 30 35 Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa 40 45 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 Xaa Xaa Xaa Cys Cys Xaa ProXaa Xaa Xaa 60 65 Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa 70 75 Xaa XaaXaa Val Xaa Leu Xaa Xaa Xaa Xaa 80 85 Xaa Met Xaa Val Xaa Xaa Cys XaaCys Xaa 90 95wherein each Xaa independently is selected from a group of one or morespecified amino acids defined as follows: “Res.” means “residue” and Xaaat res. 2=(Tyr or Lys); Xaa at res. 3=Val or Ile); Xaa at res. 4=(Ser,Asp or Glu); Xaa at res. 6=(Arg, Gin, Ser, Lys or Ala); Xaa at res.7=(Asp or Glu); Xaa at res. 8=(Leu, Val or Ile); Xaa at res. 11=(Gln,Leu, Asp, His, Asn or Ser); Xaa at res. 12=(Asp, Arg, Asn or Glu); Xaaat res. 13=(Trp or Ser); Xaa at res. 14=(Ile or Val); Xaa at res.15=(Ile or Val); Xaa at res. 16 (Ala or Ser); Xaa at res. 18=(Glu, Gin,Leu, Lys, Pro or Arg); Xaa at res. 19=(Gly or Ser); Xaa at res. 20=(Tyror Phe); Xaa at res. 21=(Ala, Ser, Asp, Met, His, Gln, Leu or Gly); Xaaat res. 23=(Tyr, Asn or Phe); Xaa at res. 26=(Glu, His, Tyr, Asp, Gln,Ala, or Ser); Xaa at res. 28=(Glu, Lys, Asp, Gin or Ala); Xaa at res.30=(Ala, Ser, Pro, Gln, Ile or Asn); Xaa at res. 31=(Phe, Leu or Tyr);Xaa at res. 33=(Leu, Val or Met); a, Thr or Pro); Xaa at res. 35=(Ser,Asp, Glu, Leu, Ala or Lys); Xaa at res. 36=(Tyr, Cys, His, Ser or Ile);Xaa at res. 37=(Met, Phe, Gly or Leu); Xaa at res. 38=(Asn, Ser or Lys);Xaa at res. 39=(Ala, Ser, Gly or Pro); Xaa at res. 40=(Thr, Leu or Ser);Xaa at res. 44=(Ile, Val or Thr); Xaa at res. 45=(Val, Leu, Met or Ile);Xaa at res. 46=(Gln or Arg); Xaa at res. 47=(Thr, Ala or Ser); Xaa atres. 48=(Leu or Ile); Xaa at res. 49=(Val or Met); Xaa at res. 50=(His,Asn or Arg); Xaa at res. 51=(Phe, Leu, Asn, Ser, Ala or Val); Xaa atres. 52=(Ile, Met, Asn, Ala, Val, Gly or Leu); Xaa at res. 0.53=(Asn,Lys, Ala, Glu, Gly or Phe); Xaa at res. 54=(Pro, Ser or Val); Xaa atres. 55=(Glu, Asp, Asn, Gly, Val, Pro or Lys); Xaa at res. 56=(Thr, Ala,Val, Lys, Asp, Tyr, Ser, Gly, Ile or His); Xaa at res. 57=(Val, Ala orIle); Xaa at res. 58=(Pro or Asp); Xaa at res. 59=(Lys, Leu or Glu); Xaaat res. 60=(Pro, Val or Ala); Xaa at res. 63=(Ala or Val); Xaa at res.65=(Thr, Ala or Glu); Xaa at res. 66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa at res. 68=(Asn, Ser, Asp or Gly); Xaa at res.69=(Ala, Pro or Ser); Xaa at res. 70=(Ile, Thr, Val or Leu); Xaa at res.71=(Ser, Ala or Pro); Xaa at res. 72=(Val, Leu, Met or Ile); Xaa at res.74=(Tyr or Phe); Xaa at res. 75=(Phe, Tyr, Leu or His); Xaa at res.76=(Asp, Asn or Leu); Xaa at res. 77=(Asp, Glu, Asn, Arg or Ser); Xaa atres. 78=(Ser, Gln, Asn, Tyr or Asp); Xaa at res. 79=(Ser, Asn, Asp, Gluor Lys); Xaa at res. 80=(Asn, Thr or Lys); Xaa at res. 82=(Ile, Val orAsn); Xaa at res. 84=(Lys or Arg); Xaa at res. 85=(Lys, Asn, Gln, His,Arg or Val); Xaa at res. 86=(Tyr, Glu or His); Xaa at res. 87=(Arg, Gln,Glu or Pro); Xaa at res. 88=(Asn, Glu, Trp or Asp); Xaa at res. 90=(Val,Thr, Ala or Ile); Xaa at res. 92=(Arg, Lys, Val, Asp, Gln or Glu); Xaaat res. 93=(Ala, Gly, Glu or Ser); Xaa at res. 95=(Gly or Ala) and Xaaat res. 97=(His or Arg).

Generic Sequence 8 (SEQ ID NO: 5) includes all of Generic Sequence 7(SEQ ID NO: 4) and in addition includes the following sequence (SEQ IDNO: 8) at its N-terminus: SEQ ID NO:8 Cys Xaa Xaa Xaa Xaa 1 5Accordingly, beginning with residue 7, each “Xaa” in Generic Sequence 8is a specified amino acid defined as for Generic Sequence 7, with thedistinction that each residue number described for Generic Sequence 7 isshifted by five in Generic Sequence 8. Thus, “Xaa at res. 2=(Tyr orLys)” in Generic Sequence 7 refers to Xaa at res. 7 in Generic Sequence8. In Generic Sequence 8, Xaa at res. 2=(Lys, Arg, Ala or Gln); Xaa atres. 3=(Lys, Arg or Met); Xaa at res. 4=(His, Arg or Gln); and Xaa atres. 5=(Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr).

In another embodiment, useful osteogenic proteins include those definedby Generic Sequences 9 and 10 (SEQ ID NOS: 6 and 7, respectively),described herein above. Specifically, Generic Sequences 9 and 10 arecomposite amino acid sequences of the following proteins: human OP-1,human OP-2, human OP-3, human BMP-2, human BMP-3, human BMP-4, humanBMP-5, human BMP-6, human BMP-8, human BMP-9, human BMP-10, humanBMP-11, Drosophila 60A, Xenopus Vg-1, sea urchin UNIVIN, human CDMP-1(mouse GDF-5), human CDMP-2 (mouse GDF-6, human BMP-13), human CDMP-3(mouse GDF-7, human BMP-12), mouse GDF-3, human GDF-1, mouse GDF-1,chicken DORSALIN, Drosophila dpp, Drosophila SCREW, mouse NODAL, mouseGDF-8, human GDF-8, mouse GDF-9, mouse GDF-10, human GDF-11, mouseGDF-11, human BMP-15, and rat BMP-3b. Like Generic Sequence 7, GenericSequence 9 accommodates the C-terminal six cysteine skeleton and, likeGeneric Sequence 8, Generic Sequence 10 accommodates the seven cysteineskeleton. Generic Sequence 9 (SEQ ID NO: 6) Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa 15 20 Xaa XaaXaa Xaa Cys Xaa Gly Xaa Cys Xaa 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 35 40 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 45 50 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 55 60 Xaa Cys Xaa Pro Xaa Xaa Xaa Xaa XaaXaa 65 70 Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 75 80 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 85 90 Xaa Xaa Xaa Cys Xaa Cys Xaa 95wherein each Xaa is independently selected from a group of one or morespecified amino acids defined as follows: “Res.” means “residue” and Xaaat res. 1=(Phe, Leu or Glu); Xaa at res. 2=(Tyr, Phe, His, Arg, Thr,Gln, Val or Glu); Xaa at res. 3=(Val, Ile, Leu or Asp); Xaa at res.4=(Ser, Asp, Glu, Asn or Phe); Xaa at res. 5=(Phe or Glu); Xaa at res.6=(Arg, Gln, Lys, Ser, Glu, Ala or Asn); Xaa at res. 7=(Asp, Glu, Leu,Ala or Gln); Xaa at res. 8=(leu, Val, Met, Ile or Phe); Xaa at res.9=(Gly, His or Lys); Xaa at res. 10=(Trp or Met); Xaa at res. 11=(Gln,Leu, His, Glu, Asn, Asp, Ser or Gly); Xaa at res. 12=(Asp, Asn, Ser,Lys, Arg, Glu or His); Xaa at res. 13=(Trp or Ser); Xaa at res. 14=(Ileor Val); Xaa at res. 15=(Ile or Val); Xaa at res. 16=(Ala, Ser, Tyr orTrp); Xaa at res. 18=(Glu, Lys, Gln, Met, Pro, Leu, Arg, His or Lys);Xaa at res. 19=(Gly, Glu, Asp, Lys, Ser, Gln, Arg or Phe); Xaa at res.20=(Tyr or Phe); Xaa at res. 21=(Ala, Ser, Gly, Met, Gln, His, Glu, Asp,Leu, Asn, Lys or Thr); Xaa at res. 22=(Ala or Pro); Xaa at res. 23=(Tyr,Phe, Asn, Ala or Arg); Xaa at res. 24=(Tyr, His, Glu, Phe or Arg); Xaaat res. 26=(Glu, Asp, Ala, Ser, Tyr, His, Lys, Arg, Gln or Gly); Xaa atres. 28=(Glu, Asp, Leu, Val, Lys, Gly, Thr, Ala or Gin); Xaa at res.30=(Ala, Ser, Ile, Asn, Pro, Glu, Asp, Phe, Gin or Leu); Xaa at res.31=(Phe, Tyr, Leu, Asn, Gly or Arg); Xaa at res. 32=(Pro, Ser, Ala orVal); Xaa at res. 33=(Leu, Met, Glu, Phe or Val); Xaa at res. 34=(Asn,Asp, Thr, Gly, Ala, Arg, Leu or Pro); Xaa at res. 35=(Ser, Ala, Glu,Asp, Thr, Leu, Lys, Gin or His); Xaa at res. 36=(Tyr, His, Cys, Ile,Arg, Asp, Asn, Lys, Ser, Glu or Gly); Xaa at res. 37=(Met, Leu, Phe,Val, Gly or Tyr); Xaa at res. 38=(Asn, Glu, Thr, Pro, Lys, His, Gly,Met, Val or Arg); Xaa at res. 39=(Ala, Ser, Gly, Pro or Phe); Xaa atres. 40=(Thr, Ser, Leu, Pro, His or Met); Xaa at res. 41=(Asn, Lys, Val,Thr or Gin); Xaa at res. 42=(His, Tyr or Lys); Xaa at res. 43=(Ala, Thr,Leu or Tyr); Xaa at res. 44=(Ile, Thr, Val, Phe, Tyr, Met or Pro); Xaaat res. 45=(Val, Leu, Met, Ile or His); Xaa at res. 46=(Gin, Arg orThr); Xaa at res. 47=(Thr, Ser, Ala, Asn or His); Xaa at res. 48=(Leu,Asn or Ile); Xaa at res. 49=(Val, Met, Leu, Pro or Ile); Xaa at res.50=(His, Asn, Arg, Lys, Tyr or Gin); Xaa at res. 51=(Phe, Leu, Ser, Asn,Met, Ala, Arg, Glu, Gly or Gin); Xaa at res. 52=(Ile, Met, Leu, Val,Lys, Gln, Ala or Tyr); Xaa at res. 53=(Asn, Phe, Lys, Glu, Asp, Ala,Gin, Gly, Leu or Val); Xaa at res. 54=(Pro, Asn, Ser, Val or Asp); Xaaat res. 55=(Glu, Asp, Asn, Lys, Arg, Ser, Gly, Thr, Gin, Pro or His);Xaa at res. 56=(Thr, His, Tyr, Ala, Ile, Lys, Asp, Ser, Gly or Arg); Xaaat res. 57=(Val, Ile, Thr, Ala, Leu or Ser); Xaa at res. 58=(Pro, Gly,Ser, Asp or Ala); Xaa at res. 59=(Lys, Leu, Pro, Ala, Ser, Glu, Arg orGly); Xaa at res. 60=(Pro, Ala, Val, Thr or Ser); Xaa at res. 61=(Cys,Val or Ser); Xaa at res. 63=(Ala, Val or Thr); Xaa at res. 65=(Thr, Ala,Glu, Val, Gly, Asp or Tyr); Xaa at res. 66=(Gin, Lys, Glu, Arg or Val);Xaa at res. 67=(Leu, Met, Thr or Tyr); Xaa at res. 68=(Asn, Ser, Gly,Thr, Asp, Glu, Lys or Val); Xaa at res. 69=(Ala, Pro, Gly or Ser); Xaaat res. 70=(Ile, Thr, Leu or Val); Xaa at res. 71=(Ser, Pro, Ala, Thr,Asn or Gly); Xaa at res. 2=(Val, Ile, Leu or Met); Xaa at res. 74=(Tyr,Phe, Arg, Thr, Tyr or Met); Xaa at res. 75=(Phe, Tyr, His, Leu, Ile,Lys, Gin or Val); Xaa at res. 76=(Asp, Leu, Asn or Glu); Xaa at res.77=(Asp, Ser, Arg, Asn, Glu, Ala, Lys, Gly or Pro); Xaa at res. 78=(Ser,Asn, Asp, Tyr, Ala, Gly, Gin, Met, Glu, Asn or Lys); Xaa at res.79=(Ser, Asn, Glu, Asp, Val, Lys, Gly, Gln or Arg); Xaa at res. 80=(Asn,Lys, Thr, Pro, Val, Ile, Arg, Ser or Gln); Xaa at res. 81=(Val, Ile, Thror Ala); Xaa at res. 82=(Ile, Asn, Val, Leu, Tyr, Asp or Ala); Xaa atres. 83=(Leu, Tyr, Lys or Ile); Xaa at res. 84=(Lys, Arg, Asn, Tyr, Phe,Thr, Glu or Gly); Xaa at res. 85=(Lys, Arg, His, Gln, Asn, Glu or Val);Xaa at res. 86=(Tyr, His, Glu or Ile); Xaa at res. 87=(Arg, Glu, Gln,Pro or Lys); Xaa at res. 88=(Asn, Asp, Ala, Glu, Gly or Lys); Xaa atres. 89=(Met or Ala); Xaa at res. 90=(Val, Ile, Ala, Thr, Ser or Lys);Xaa at res. 91=(Val or Ala); Xaa at res. 92=(Arg, Lys, Gln, Asp, Glu,Val, Ala, Ser or Thr); Xaa at res. 93=(Ala, Ser, Glu, Gly, Arg or Thr);Xaa at res. 95=(Gly, Ala or Thr); Xaa at res. 97=(His, Arg, Gly, Leu orSer). Further, after res. 53 in rBMP-3b and mGDF-10 there is an Ile;after res. 54 in GDF-1 there is a T; after res. 54 in BMP-3 there is aV; after res. 78 in BMP-8 and Dorsalin there is a G; after res. 37 inhGDF-1 there is Pro, Gly, Gly, Pro.

Generic Sequence 10 (SEQ ID NO: 7) includes all of Generic Sequence 9(SEQ ID NO: 6) and in addition includes the following sequence (SEQ IDNO: 9) at its N-terminus: SEQ ID NO: 9 Cys Xaa Xaa Xaa Xaa 1 5Accordingly, beginning with residue 6, each “Xaa” in Generic Sequence 10is a specified amino acid defined as for Generic Sequence 9, with thedistinction that each residue number described for Generic Sequence 9 isshifted by five in Generic Sequence 10. Thus, “Xaa at res. 1=(Tyr, Phe,His, Arg, Thr, Lys, Gln, Val or Glu)” in Generic Sequence 9 refers toXaa at res. 6 in Generic Sequence 10. In Generic Sequence 10, Xaa atres. 2=(Lys, Arg, Gln, Ser, His, Glu, Ala, or Cys); Xaa at res. 3=(Lys,Arg, Met, Lys, Thr, Leu, Tyr, or Ala); Xaa at res. 4=(His, Gln, Arg,Lys, Thr, Leu, Val, Pro, or Tyr); and Xaa at res. 5=(Gln, Thr, His, Arg,Pro, Ser, Ala, Gln, Asn, Tyr, Lys, Asp, or Leu).

Based upon alignment of the naturally-occurring morphogens within thedefinition of Generic Sequence 10, it should be clear that gaps and/orinsertions of one or more amino acid residues can be tolerated (withoutabrogating or substantially impairing biological activity) at leastbetween or involving residues 11-12, 42-43, 59-60, 68-69 and 83-84.

As noted above, certain preferred morphogenic polypeptide sequencesuseful in this invention have greater than 60% identity, preferablygreater than 65% identity, with the amino acid sequence defining thepreferred reference sequence of hOP-1. These particularly preferredsequences include allelic and phylogenetic counterpart variants of theOP-1 and OP-2 proteins, including the Drosophila 60A protein, as well asthe closely related proteins BMP-5, BMP-6 and Vgr-1. Accordingly, incertain particularly preferred embodiments, useful morphogenic proteinsinclude active proteins comprising pairs of polypeptide chains withinthe generic amino acid sequence herein referred to as “OPX” (SEQ ID NO:3), which defines the seven cysteine skeleton and accommodates thehomologies between several identified variants of OP-1 and OP-2.Accordingly, each “Xaa” at a given position in OPX independently isselected from the residues occurring at the corresponding position inthe C-terminal sequence of mouse or human OP-1 or OP-2. Specifically,each “Xaa” is independently selected from a group of one or morespecified amino acids as defined below: Cys Xaa Xaa His Glu Leu Tyr ValSer Phe Xaa Asp Leu Gly Trp 1 5 10 15 Xaa Asp Trp Xaa Ile Ala Pro XaaGly Tyr Xaa Ala Tyr Tyr Cys 20 25 30 Glu Gly Glu Cys Xaa Phe Pro Leu XaaSer Xaa Met Asn Ala Thr 35 40 45 Asn His Ala Ile Xaa Gln Xaa Leu Val HisXaa Xaa Xaa Pro Xaa 50 55 60 Xaa Val Pro Lys Xaa Cys Cys Ala Pro Thr XaaLeu Xaa Ala Xaa 65 70 75 Ser Val Leu Tyr Xaa Asp Xaa Ser Xaa Asn Val IleLeu Xaa Lys 80 85 90 Xaa Arg Asn Met Val Val Xaa Ala Cys Gly Cys His 95100wherein Xaa at res. 2=(Lys or Arg); Xaa at res. 3 (Lys or Arg); Xaa atres. 11=(Arg or Gln); Xaa at res. 16=(Gin or Leu); Xaa at res. 19=(Ileor Val); Xaa at res. 23=(Glu or Gln); Xaa at res. 26=(Ala or Ser); Xaaat res. 35=(Ala or Ser); Xaa at res. 39=(Asn or Asp); Xaa at res.41=(Tyr or Cys); Xaa at res. 50=(Val or Leu); Xaa at res. 52=(Ser orThr); Xaa at res. 56=(Phe or Leu); Xaa at res. 57=(Ile or Met); Xaa atres. 58=(Asn or Lys); Xaa at res. 60=(Glu, Asp or Asn); Xaa at res.61=(Thr, Ala or Val); Xaa at res. 65=(Pro or Ala); Xaa at res. 71=(Glnor Lys); Xaa at res. 73=(Asn or Ser); Xaa at res. 75=(Ile or Thr); Xaaat res. 80=(Phe or Tyr); Xaa at res. 82=(Asp or Ser); Xaa at res.84=(Ser or Asn); Xaa at res. 89=(Lys or Arg); Xaa at res. 91=(Tyr orHis); and Xaa at res. 97=(Arg or Lys).

In still another preferred embodiment, useful morphogenically-activeproteins have polypeptide chains with amino acid sequences comprising asequence encoded by a nucleic acid that hybridizes with DNA or RNAencoding reference morphogen sequences, e.g., C-terminal sequencesdefining the conserved seven cysteine skeletons of OP-1, OP-2, BMP-2,BMP-4, BMP-5, BMP-6, 60A, GDF-3, GDF-5, GDF-6, GDF-7 and the like. Asused herein, high stringency hybridization conditions are defined ashybridization according to known techniques in 40% formamide, 5×SSPE, 5×Denhardt's Solution, and 0.1% SDS at 37° C. overnight, and washing in0.1×SSPE, 0.1% SDS at 50° C. Standard stringency conditions are wellcharacterized in standard molecular biology cloning texts. See, forexample, MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed., ed. bySambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press:1989); DNA CLONING, Volumes I and II (D. N. Glover ed., 1985);OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait ed., 1984); NUCLEIC ACIDHYBRIDIZATION (B. D. Hames & S. J. Higgins eds. 1984); and B. Perbal, APRACTICAL GUIDE TO MOLECULAR CLONING (1984).

In other embodiments, as an alternative to the administration of amorphogenic protein, an effective amount of an agent competent tostimulate or induce increased endogenous morphogen expression in amammal may be administered by any of the routes described herein. Such amorphogen inducer may be provided to a mammal, e.g., by systemicadministration to the mammal or by direct administration to the neuraltissue. A method for identifying and testing inducers (stimulatingagents) competent to modulate the levels of endogenous morphogens in agiven tissue is described in published applications WO93/05172 andWO93/05751, each of which is incorporated by reference herein. Briefly,candidate compounds are identified and tested by incubation in vitrowith test tissue or cells, or a cultured cell line derived therefrom,for a time sufficient to allow the compound to affect the production,i.e., cause the expression and/or secretion, of a morphogen produced bythe cells of that tissue. Suitable tissue, or cultured cells of asuitable tissue, are preferably selected from renal epithelium, ovariantissue, fibroblasts, and osteoblasts.

In yet other embodiments, an agent which acts as an agonist of amorphogen receptor may be administered instead of the morphogen itself.Such an agent may also be referred to an a morphogen “mimic,” “mimetic,”or “analog.” Thus, for example, a small peptide or other molecule whichcan mimic the activity of a morphogen in binding to and activating themorphogen's receptor may be employed as an equivalent of the morphogen.Preferably the agonist is a full agonist, but partial morphogen receptoragonists may also be advantageously employed. Methods of identifyingsuch agonists are known in the art and include assays for compoundswhich induce morphogen-mediated responses (e.g., induction ofdifferentiation of metanephric mesenchyme, induction of endochondralbone formation). For example, methods of identifying morphogen inducersor agonists of morphogen receptors may be found in U.S. Ser. No.08/478,097 filed Jun. 7, 1995 and U.S. Ser. No. 08/507,598 filed Jul.26, 1995, disclosures of which are incorporated herein by reference.

As a general matter, methods of the present invention may be applied tothe treatment of any mammalian subject at risk of or afflicted with aneural tissue insult or neuropathy. The invention is suitable for thetreatment of any primate, preferably a higher primate such as a human.In addition, however, the invention may be employed in the treatment ofdomesticated mammals which are maintained as human companions (e.g.,dogs, cats, horses), which have significant commercial value (e.g.,goats, pigs, sheep, cattle, sporting or draft animals), which havesignificant scientific value (e.g., captive or free specimens ofendangered species, or inbred or engineered animal strains), or whichotherwise have value.

C. Formulations and Methods of Treatment

Morphogens, morphogen inducers, or agonists of morphogen receptors ofthe present invention may be administered by any route which iscompatible with the particular morphogen, inducer, or agonist employed.Thus, as appropriate, administration may be oral or parenteral,including intravenous and intraperitoneal routes of administration. Inaddition, administration may be by periodic injections of a bolus of themorphogen, inducer or agonist, or may be made more continuous byintravenous or intraperitoneal administration from a reservoir which isexternal (e.g., an i.v. bag) or internal (e.g., a bioerodable implant,or a colony of implanted, morphogen-producing cells).

Therapeutic agents of the invention (i.e., morphogens, morphogeninducers or agonists of morphogen receptors) may be provided to anindividual by any suitable means, directly (e.g., locally, as byinjection, implantation or topical administration to a tissue locus) orsystemically (e.g., parenterally or orally). Where the agent is to beprovided parenterally, such as by intravenous, subcutaneous,intramolecular, ophthalmic, intraperitoneal, intramuscular, buccal,rectal, vaginal, intraorbital, intracerebral, intracranial, intraspinal,intraventricular, intrathecal, intracisternal, intracapsular, intranasalor by aerosol administration, the agent preferably comprises part of anaqueous or physiologically compatible fluid suspension or solution.Thus, the morphogen carrier or vehicle is physiologically acceptable sothat in addition to delivery of the desired agent to the patient, itdoes not otherwise adversely affect the patient's electrolyte and/orvolume balance. The fluid medium for the agent thus can comprise normalphysiologic saline (e.g., 9.85% aqueous NaCl, 0.15M, pH 7-7.4).

Association of the mature morphogen dimer with a morphogen pro domainresults in the pro form of the morphogen which typically is more solublein physiological solutions than the corresponding mature form. In fact,endogenous morphogens are thought to be transported (e.g., secreted andcirculated) in the mammalian body in this form. This soluble form of theprotein can be obtained from culture medium of morphogen-secretingmammalian cells, e.g., cells transfected with nucleic acid encoding andcompetent to express the morphogen. Alternatively, a soluble species canbe formulated by complexing the mature, morphogenically-activepolypeptide dimer (or an active fragment thereof) with a morphogen prodomain polypeptide or a solubility-enhancing fragment thereof.Solubility-enhancing pro domain fragments can be any N-terminal,C-terminal or internal fragment of the pro region of a member of themorphogen family that complexes with the mature polypeptide dimer toenhance stability and/or dissolubility of the resulting noncovalent orconvalent complex. Typically, useful fragments are those cleaved at theproteolytic site Arg-Xaa-Xaa-Arg. A detailed description of solublecomplex forms of morphogenic proteins, including how to make, test anduse them, is described in WO 94/03600 (PCT US 93/07189). In the case ofOP-1, useful pro domain polypeptide fragments include the intact prodomain polypeptide (residues 30-292) and fragments 48-292 and 158-292,all of SEQ ID NO: 2. Another molecule capable of enhancing solubilityand particularly useful for oral administrations, is casein. Forexample, addition of 0.2% casein increases solubility of the matureactive form of OP-1 by 80%. Other components found in milk and/orvarious serum proteins may also be useful.

Useful solutions for parenteral administration may be prepared by any ofthe methods well known in the pharmaceutical art, described, forexample, in REMINGTON'S PHARMACEUTICAL SCIENCES (Gennaro, A., ed.), MackPub., 1990. Formulations of the therapeutic agents of the invention mayinclude, for example, polyalkylene glycols such as polyethylene glycol,oils of vegetable origin, hydrogenated naphthalenes, and the like.Formulations for direct administration, in particular, may includeglycerol and other compositions of high viscosity to help maintain theagent at the desired locus. Biocompatible, preferably bioresorbable,polymers, including, for example, hyaluronic acid, collagen, tricalciumphosphate, polybutyrate, lactide, and glycolide polymers andlactide/glycolide copolymers, may be useful excipients to control therelease of the agent in vivo. Other potentially useful parenteraldelivery systems for these agents include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, andliposomes. Formulations for inhalation administration contain asexcipients, for example, lactose, or may be aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycocholateand deoxycholate, or oily solutions for administration in the form ofnasal drops, or as a gel to be applied intranasally. Formulations forparenteral administration may also include glycocholate for buccaladministration, methoxysalicylate for rectal administration, or cutricacid for vaginal administration. Suppositories for rectal administrationmay also be prepared by mixing the morphogen, inducer or agonist with anon-irritating excipient such as cocoa butter or other compositionswhich are solid at room temperature and liquid at body temperatures.

Formulations for topical administration to the skin surface may beprepared by dispersing the morphogen, inducer or agonist with adermatologically acceptable carrier such as a lotion, cream, ointment orsoap. Particularly useful are carriers capable of forming a film orlayer over the skin to localize application and inhibit removal. Fortopical administration to internal tissue surfaces, the agent may bedispersed in a liquid tissue adhesive or other substance known toenhance adsorption to a tissue surface. For example,hydroxypropylcellulose or fibrinogen/thrombin solutions may be used toadvantage. Alternatively, tissue-coating solutions, such aspectin-containing formulations may be used.

Alternatively, the agents described herein may be administered orally.Oral administration of proteins as therapeutics generally is notpracticed, as most proteins are readily degraded by digestive enzymesand acids in the mammalian digestive system before they can be absorbedinto the bloodstream. However, the morphogens described herein typicallyare acid stable and protease-resistant (see, for example, U.S. Pat. No.4,968,590). In addition, at least one morphogen, OP-1, has beenidentified in mammary gland extract, colostrum and 57-day milk.Moreover, the OP-1 purified from mammary gland extract ismorphogenically-active and is also detected in the bloodstream. Maternaladministration, via ingested milk, may be a natural delivery route ofTGF-β superfamily proteins. Letterio, et al., Science 264: 1936-1938(1994), report that TGF-β is present in murine milk, and thatradiolabelled TGF-β is absorbed by gastrointestinal mucosa of sucklingjuveniles. Labeled, ingested TGF-β appears rapidly in intact form in thejuveniles' body tissues, including lung, heart and liver. Finally,soluble form morphogen, e.g., mature morphogen associated with the prodomain, is morphogenically-active. These findings, as well as thosedisclosed in the examples below, indicate that oral and parenteraladministration are viable means for administering TGF-β superfamilyproteins, including the morphogens, to an individual. In addition, whilethe mature forms of certain morphogens described herein typically aresparingly soluble, the morphogen form found in milk (and mammary glandextract and colostrum) is readily soluble, probably by association ofthe mature, morphogenically-active form with part or all of the prodomain of the expressed, full length polypeptide sequence and/or byassociation with one or more milk components. Accordingly, the compoundsprovided herein may also be associated with molecules capable ofenhancing their solubility in vitro or in vivo.

Where the morphogen is intended for use as a therapeutic for disordersof the CNS, an additional problem must be addressed: overcoming theblood-brain barrier, the brain capillary wall structure that effectivelyscreens out all but selected categories of substances present in theblood, preventing their passage into the brain. The blood-brain barriercan be bypassed effectively by direct infusion of the morphogen ormorphogen-stimulating agent into the brain, or by intranasaladministration or inhalation of formulations suitable for uptake andretrograde transport by olfactory neurons. Alternatively, the morphogenor morphogen-stimulating agent can be modified to enhance its transportacross the blood-brain barrier. For example, truncated forms of themorphogen or a morphogen-stimulating agent may be most successful.Alternatively, the morphogens, inducers or agonists provided herein canbe derivatized or conjugated to a lipophilic moiety or to a substancethat is actively transported across the blood-brain barrier, usingstandard means known to those skilled in the art. See, for example,Pardridge, Endocrine Reviews 7: 314-330 (1986) and U.S. Pat. No.4,801,575.

The compounds provided herein may also be associated with moleculescapable of targeting the morphogen, inducer or agonist to the desiredtissue. For example, an antibody, antibody fragment, or other bindingprotein that interacts specifically with a surface molecule on cells ofthe desired tissue, may be used. Useful targeting molecules may bedesigned, for example, using the single chain binding site technologydisclosed in U.S. Pat. No. 5,091,513. Targeting molecules can becovalently or non-covalently associated with the morphogen, inducer oragonist.

As will be appreciated by one of ordinary skill in the art, theformulated compositions contain therapeutically-effective amounts of themorphogen, morphogen inducers or agonists of morphogen receptors. Thatis, they contain an amount which provides appropriate concentrations ofthe agent to the affected nervous system tissue for a time sufficient tostimulate a detectable restoration of impaired central or peripheralnervous system function, up to and including a complete restorationthereof. As will be appreciated by those skilled in the art, theseconcentrations will vary depending upon a number of factors, includingthe biological efficacy of the selected agent, the chemicalcharacteristics (e.g., hydrophobicity) of the specific agent, theformulation thereof, including a mixture with one or more excipients,the administration route, and the treatment envisioned, includingwhether the active ingredient will be administered directly into atissue site, or whether it will be administered systemically. Thepreferred dosage to be administered is also likely to depend onvariables such as the condition of the diseased or damaged tissues, andthe overall health status of the particular mammal. As a general matter,single, daily, biweekly or weekly dosages of 0.00001-1000 mg of amorphogen are sufficient, with 0.0001-100 mg being preferable, and 0.001to 10 mg being even more preferable. Alternatively, a single, daily,biweekly or weekly dosage of 0.01-1000 μg/kg body weight, morepreferably 0.01-10 mg/kg body weight, may be advantageously employed.The present effective dose can be administered in a single dose or in aplurality (two or more) of installment doses, as desired or consideredappropriate under the specific circumstances. A bolus injection ordiffusable infusion formulation can be used. If desired to facilitaterepeated or frequent infusions, implantation of a semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular)may be advisable.

The morphogens, inducers or agonists of the invention may, of course, beadministered alone or in combination with other molecules known to bebeneficial in the treatment of the conditions described herein. Forexample, various well-known growth factors, hormones, enzymes,therapeutic compositions, antibiotics, or other bioactive agents canalso be administered with the morphogen. Thus, various known growthfactors such as NGF, EGF, PDGF, IGF, FGF, TGF-α, and TGF-β, as well asenzymes, enzyme inhibitors, antioxidants, anti-inflammatory agents, freeradical scavenging agents, antibiotics and/orchemoattractant/chemotactic factors, can be included in the presentmorphogen formulation.

EXAMPLE 1 Preparation of Soluble Morphogen Protein Solutions for In vivoAdministration

A. Aqueous Solutions

While the mature dimeric morphogenic proteins defined herein aretypically sparingly soluble in physiological buffers, they can besolubilized to form injectable suspensions or solutions. One exemplaryaqueous formulation containing a morphogen is made, for example, bydispersing the morphogen in 50% ethanol containing acetonitrile in 0.1%trifluoroacetic acid (TFA) or 0.1% HCl, or in an equivalent solvent. Onevolume of the resultant solution then is added, for example, to tenvolumes of phosphate buffered saline (PBS), which further may include0.1-0.2% human serum albumin (HSA) or a similar carrier protein. Theresultant solution is preferably vortexed extensively to produce aphysiologically acceptable morphogen formulation.

In another embodiment, the morphogen, including OP-1, is solubilized byreducing the pH of the solution. In one preferred formulation, theprotein is solubilized in 0.2 mM acetate buffer, pH 4.5, containing 5%mannitol, to render the solution more isotonic. Other standard means forcreating physiologically acceptable formulations are within the skill ofthe art.

B. Soluble Complex Formulations

Another preferred form is a dimeric morphogenic protein comprising atleast the C-terminal seven cysteine skeleton characteristic of themorphogen family, complexed with a peptide comprising a pro region of amember of the morphogen family, or a solubility-enhancing fragmentthereof, or an allelic, phylogenetic or other sequence variant thereof.The solubility-enhancing fragment is any N-terminal or C-terminalfragment of the pro domain polypeptide of a member of the morphogenfamily that complexes with the mature polypeptide dimer to enhance thestability of the resulting soluble complex. Preferably, the dimericmorphogenic protein is complexed with two such pro domain peptides.

As described above and in published application WO 94/03600,incorporated by reference herein, the soluble complex form is isolatedfrom the cell culture media (or a body fluid) under appropriateconditions. Alternatively, the complex is formulated in vitro.

Soluble morphogen complexes are isolated from conditioned media using asimple, three step chromatographic protocol performed in the absence ofdenaturants. The protocol involves running the media (or body fluid)over an affinity column, followed by ion exchange and gel filtrationchromatographies generally as described in WO 94/03600. The affinitycolumn described below is a Zn-IMAC column. The present example useshuman OP-1, and is not intended to be limiting. The present protocol hasgeneral applicability to the purification of a variety of morphogens,all of which are anticipated to be isolatable using only minormodifications of the protocol described below. An alternative protocolalso envisioned to have utility includes an immunoaffinity column,created using standard procedures and, for example, using antibodyspecific for a given morphogen pro domain (complexed, for example, to aprotein. A-conjugated Sepharose column). Protocols for developingimmunoaffinity columns are well described in the art (see, for example,GUIDE TO PROTEIN PURIFICATION, M. Deutscher, ed., Academic Press, SanDiego, 1990, particularly sections VII and XI thereof).

In this example, OP-1 was expressed in mammalian (CHO, Chinese hamsterovary) cells as described in the art (see, for example, internationalapplication US90/05903 (WO 91/05802). The CHO cell conditioned mediacontaining 0.5% FBS is initially purified using Immobilized Metal-IonAffinity Chromatography (IMAC). The soluble OP-1 complex fromconditioned media binds very selectively to the Zn-IMAC resin and a highconcentration of imidazole (50 mM imidazole, pH 8.0) is required for theeffective elution of the bound complex. The Zn-IMAC purified solubleOP-1 is next applied to an S-Sepharose action-exchange columnequilibrated in 20 mM NaPO₄ (pH 7.0) with 50 mM NaCl. The protein thenis applied to a Sephacryl S-200HR column equilibrated in TBS. Usingsubstantially the same protocol, soluble morphogens can also be isolatedfrom one or more body fluids, including milk, serum, cerebrospinal fluidor peritoneal fluid.

The soluble OP-1 complex elutes with an apparent molecular weight of 110kDa. This agrees well with the predicted composition of the soluble OP-1complex, with one mature OP-1 dimer (35-36 kDa) associated with two prodomains (39 kDa each). Purity of the final complex can be verified byrunning the appropriate fraction in a reduced 15% polyacrylamide gel.

As an alternative to purifying soluble complexes from culture media or abody fluid, soluble complexes can be formulated from purified prodomains and mature dimeric species. Successful complex formationapparently requires association of the components under denaturingconditions sufficient to relax the folded structure of these molecules,without affecting disulfide bonds. Preferably, the denaturing conditionsmimic the environment of an intracellular vesicle sufficiently such thatthe cleaved pro domain polypeptide has an opportunity to associate withthe mature dimeric protein under relaxed folding conditions. Theconcentration of denaturant in the solution then is decreased in acontrolled, preferably step-wise manner, so as to allow proper refoldingof the dimer and pro domain peptides, while maintaining the associationof the pro domain peptides with the mature dimer. Useful denaturantsinclude 4-6 M urea or guanidine hydrochloride (GuHCl), in bufferedsolutions of pH 4-10, preferably pH 6-8. The soluble complex then isformed by controlled dialysis or dilution into a solution having a finaldenaturant concentration of less than 0.1-2M urea or GuHCl, preferably1-2 M urea or GuHCl, which then preferably can be diluted into aphysiological buffer. Protein purification/renaturing procedures andconsiderations are well described in the art, and details for developinga suitable renaturing protocol readily can be determined by one havingordinary skill in the art. One useful text on the subject is GUIDE TOPROTEIN PURIFICATION, M. Deutscher, ed., Academic Press, San Diego,1990, particularly section V. Complex formation may also be aided byaddition of one or more chaperone proteins.

The stability of the highly purified soluble morphogen complex in aphysiological buffer, e.g., Tris-buffered saline (TBS) andphosphate-buffered saline (PBS), can be enhanced by any of a number ofmeans, including any one or more of three classes of additives. Theseadditives include basic amino acids (e.g., L-arginine, lysine andbetaine); nonionic detergents (e.g., Tween 80 or NonIdet P-120); andcarrier proteins (e.g., serum albumin and casein). Useful concentrationsof these additives include 1-100 mM, preferably 10-70 mM, including 50mM, basic amino acid; 0.01-1.0%, preferably 0.05-0.2%, including 0.1%(v/v) nonionic detergent; and 0.01-1.0%, preferably 0.05-0.2%, including0.1% (w/v) carrier protein.

EXAMPLE 2 Identification of Morphogen-Expressing Tissue

Determining the tissue distribution of morphogens may be used toidentify different morphogens expressed in a given tissue, as well as toidentify new, related morphogens. Tissue distribution also may be usedto identify useful morphogen-producing tissue for use in screening andidentifying candidate morphogen-stimulating agents. The morphogens (ortheir mRNA transcripts) readily are identified in different tissuesusing standard methodologies and minor modifications thereof in tissueswhere expression may be low. For example, protein distribution may bedetermined using standard Western blot analysis or immunofluorescenttechniques, and antibodies specific to the morphogen or morphogens ofinterest. Similarly, the distribution of morphogen transcripts may bedetermined using standard Northern hybridization protocols andtranscript-specific probes.

Any probe capable of hybridizing specifically to a transcript, anddistinguishing the transcript of interest from other, relatedtranscripts may be used. Because the morphogens described herein sharesuch high sequence homology in their active, C-terminal domains, thetissue distribution of a specific morphogen transcript may best bedetermined using a probe specific for the pro region of the immatureprotein and/or the N-terminal region of the mature protein. Anotheruseful sequence is the 3′ non-coding region flanking and immediatelyfollowing the stop codon. These portions of the sequence varysubstantially among the morphogens of this invention, and accordingly,are specific for each protein. For example, a particularly usefulVgr-1-specific probe sequence is the PvuII-SacI fragment, a 265 bpfragment encoding both a portion of the untranslated pro region and theN-terminus of the mature sequence. See Lyons, et al., PNAS 86: 4554-4558(1989) for a description of the cDNA sequence. Similarly, particularlyuseful mOP-1-specific probe sequences are the BstX1-BglI fragment, a0.68 Kb sequence that covers approximately two-thirds of the mOP-1 proregion; a StuI-StuI fragment, a 0.2 Kb sequence immediately upstream ofthe 7-cysteine domain; and the Earl-Pst1 fragment, an 0.3 Kb fragmentcontaining a portion of the 3′ untranslated sequence (See SEQ ID NO: 18,where the pro region is defined essentially by residues 30-291.) Similarapproaches may be used, for example, with hOP-1 (SEQ ID NO: 16) or humanor mouse OP-2 (SEQ ID NOS: 20 and 22.)

Using these morphogen-specific probes, which may be syntheticallyengineered or obtained from cloned sequences, morphogen transcripts canbe identified in mammalian tissue, using standard methodologies wellknown to those having ordinary skill in the art. Briefly, total RNA isprepared from various adult murine tissues (e.g., liver, kidney, testis,heart, brain, thymus and stomach) by a standard methodology such as bythe method of Chomczyaski, et al., Anal. Biochem 162: 156-159 (1987) anddescribed below. Poly (A)+ RNA is prepared by using oligo (dT)-cellulosechromatography (e.g., Type 7, from Pharmacia LKB Biotechnology, Inc.).Poly (A)+ RNA (generally 15 mg) from each tissue is fractionated on a 1%agarose/formaldehyde gel and transferred onto a Nytran membrane(Schleicher & Schuell). Following the transfer, the membrane is baked at80° C. and the RNA is cross-linked under UV light (generally 30 secondsat 1 mW/cm²). Prior to hybridization, the appropriate probe is denaturedby heating. The hybridization is carried out in a lucite cylinderrotating in a roller bottle apparatus at approximately 1 rev/min forapproximately 15 hours at 37° C. using a hybridization mix of 40%formamide, 5× Denhardts, 5×SSPE, and 0.1% SDS. Following hybridization,the non-specific counts are washed off the filters in 0.1×SSPE, 0.1% SDSat 50° C.

Examples demonstrating the tissue distribution of various morphogens,including Vgr-1, OP-1, BMP2, BMP3, BMP4, BMP5, GDF-1, and OP-2 indeveloping and adult tissue are disclosed in co-pending U.S. Ser. No.752,764, and in Ozkaynak, et al., Biochem. Biophys. Res. Comm. 179:116-123 (1991), and Ozkaynak, et al., (JBC, in press) (1992), thedisclosures of which are incorporated herein by reference. Using thegeneral probing methodology described herein, northern blothybridizations using probes specific for these morphogens to probebrain, spleen, lung, heart, liver and kidney tissue indicate thatkidney-related tissue appears to be the primary expression source forOP-1, with brain, heart and lung tissues being secondary sources. Lungtissue appears to be the primary tissue expression source for Vgr-1,BMP5, BMP4 and BMP3. Lower levels of Vgr-1 also are seen in kidney andheart tissue, while the liver appears to be a secondary expressionsource for BMP5, and the spleen appears to be a secondary expressionsource for BMP4. GDF-1 appears to be expressed primarily in braintissue. To date, OP-2 appears to be expressed primarily in earlyembryonic tissue. Specifically, northern blots of murine embryos and6-day post-natal animals shows abundant OP2 expression in 8-day embryos.Expression is reduced significantly in 17-day embryos and is notdetected in post-natal animals.

EXAMPLE 3 Morphogen Localization in the Nervous System

Morphogens have been identified in developing and adult rat brain andspinal cord tissue, as determined both by northern blot hybridization ofmorphogen-specific probes to mRNA extracts from developing and adultnerve tissue (see Example 2, above) and by immunolocalization studies.For example, northern blot analysis of developing rat tissue hasidentified significant OP-1 mRNA transcript expression in the CNS (U.S.Ser. No. 752,764, and Ozkaynak, et al., Biochem. Biophys. Res. Comm.,179: 11623 (1991) and Ozkaynak, et al., JBC, in press (1992)). GDF-1mRNA appears to be expressed primarily in developing and adult nervetissue, specifically in the brain, including the cerebellum and brainstem, spinal cord and peripheral nerves. Lee, S., PNAS 88: 4250-4254(1991). BMP2B (also referred in the art as BMP4) and Vgr-1 transcriptsalso have been reported to be expressed in nerve tissue (Jones, et al.,Development 111: 531-542 (1991)), although the nerve tissue does notappear to be the primary expression tissue for these genes. Ozkaynak, etal., JBC in press (1992). Specifically, CBMP2 transcripts are reportedin the region of the diencephalon associated with pituitary development,and Vgr-1 transcripts are reported in the anteroposterior axis of theCNS, including in the roof plate of the developing neural tube, as wellas in the cells immediately adjacent the floor plate of the developingneural tube. In older rats, Vgr-1 transcripts are reported in developinghippocampus tissue. In addition, the genes encoding OP-1 and BMP2originally were identified by probing human hippocampus cDNA libraries.

Immunolocalization studies, performed using standard methodologies knownin the art and disclosed in U.S. Ser. No. 752,764, filed Aug. 30, 1991,the disclosure of which is incorporated herein, localized OP-1expression to particular areas of developing and adult rat brain andspinal cord tissue. Specifically, OP-1 protein expression was assessedin adult (2-3 months old) and five or six-day old mouse embryonic nervetissue, using standard morphogen-specific antisera, specifically, rabbitanti-OP-1 antisera, made using standard antibody protocols known in theart and preferably purified on an OP-1 affinity column. The antibodyitself was labelled using standard fluorescent labelling techniques, ora labelled anti-rabbit IgG molecule was used to visualize bound OP-1antibody.

As can be seen in FIG. 2, immunofluorescence staining demonstrates thepresence of OP-1 in adult rat central nervous system (CNS.) Similar andextensive staining is seen in both the brain (Panel 1A) and spinal cord(Panel 1B). OP-1 appears to be localized predominantly to theextracellular matrix of the grey matter (neuronal cell bodies),distinctly present in all areas except the cell bodies themselves. Inwhite matter, formed mainly of myelinated nerve fibers, staining appearsto be confined to astrocytes (glial cells). A similar staining patternalso was seen in newborn rat (10 day old) brain sections.

In addition, OP-1 has been specifically localized in the substantianigra, which is composed primarily of striatal basal ganglia, a systemof accessory motor neurons that function is association with thecerebral cortex and corticospinal system. Dysfunctions in thissubpopulation or system of neurons are associated with a number ofneuropathies, including Huntington's chorea and Parkinson's disease.

OP-1 also has been localized at adendema glial cells, known to secretefactors into the cerebrospinal fluid, and which occur around theintraventricular valve, coroid fissure, and central canal of the brainin both developing and adult rat.

Finally, morphogen inhibition in developing embryos inhibits nervetissue development. Specifically, 9-day mouse embryo cells, cultured invitro under standard culturing conditions, were incubated in thepresence and absence of an OP-1-specific monoclonal antibody preparedusing recombinantly produced, purified mature OP-1 and the immunogen.The antibody was prepared using standard antibody production means wellknown in the art and as described generally in Example 14. After twodays, the effect of the antibody on the developing embryo was evaluatedby histology. As determined by histological examination, theOP-1-specific antibody specifically inhibits eye lobe formation in thedeveloping embryo. In particular, the diencephalon outgrowth does notdevelop. In addition, the heart is malformed and enlarged. Moreover, inseparate immunolocalization studies on embryo sections with labelledOP-1 specific antibody, the OP-1-specific antibody localizes to neuralepithelia.

The endogenous morphogens which act on neuronal cells may be expressedand secreted by nerve tissue cells, e.g., by neurons and/or glial cellsassociated with the neurons, and/or they may be transported to theneurons by the cerebrospinal fluid and/or bloodstream. Recently, OP-1has been identified in the human blood (See Example 10, below). Inaddition, transplanted Schwann cells recently have been shown tostimulate nerve fiber formation in rat spinal cord, including inducingvascularization and myelin sheath formation around at least some of thenew neuronal processes. Bunge, Exp. Neurology 114: 254-257 (1991). Theregenerative property of these cells may be mediated by the secretion ofa morphogen by the Schwann cells.

EXAMPLE 4 Morphogen Enhancement of Neuronal Cell Survival

The morphogens described herein enhance cell survival, particularly ofneuronal cells at risk of dying. For example, fully differentiatedneurons are non-mitotic and die in vitro when cultured under standardmammalian cell culture conditions, using a chemically defined or lowserum medium known in the art. See, for example, Charness, J. Biol.Chem. 26: 3164-3169 (1986) and Freese, et al., Brain Res. 521: 254-264(1990). However, if a primary culture of non-mitotic neuronal cells istreated with a morphogen, the survival of these cells is enhancedsignificantly. For example, a primary culture of striatal basal gangliaisolated from the substantia nigra of adult rat brain was prepared usingstandard procedures, e.g., by dissociation by trituration with pasteurpipette of substantia nigra tissue, using standard tissue culturingprotocols, and grown in a low serum medium, e.g., containing 50% DMEM(Dulbecco's modified Eagle's medium), 50% F-12 medium, heat inactivatedhorse serum supplemented with penicillin/streptomycin and 4 g/l glucose.Under standard culture conditions, these cells are undergoingsignificant cell death by three weeks when cultured in a serum-freemedium. Cell death is evidenced morphologically by the inability ofcells to remain adherent and by changes in their ultrastructuralcharacteristics, e.g., by chromatin clumping and organelledisintegration.

In this example, the cultured basal ganglia were treated with chemicallydefined medium conditioned with 0.1-100 ng/ml OP-1. Fresh,morphogen-conditioned medium was provided to the cells every 3-4 days.Cell survival was enhanced significantly and was dose dependent upon thelevel of OP-1 added: cell death decreased significantly as theconcentration of OP-1 was increased in cell cultures. Specifically,cells remained adherent and continued to maintain the morphology ofviable differentiated neurons. In the absence of morphogen treatment,the majority of the cultured cells dissociated and underwent cellnecrosis.

Dysfunctions in the basal ganglia of the substantia nigra are associatedwith Huntington's chorea and parkinsonism in vivo. The ability of themorphogens defined herein to enhance neuron survival indicates thatthese morphogens will be useful as part of a therapy to enhance survivalof neuronal cells at risk of dying in vivo due, for example, to aneuropathy or chemical or mechanical trauma. It is particularlyanticipated that these morphogens will provide a useful therapeuticagent to treat neuropathies which affect the striatal basal ganglia,including Huntington's chorea and Parkinson's disease. For clinicalapplications, the morphogen may be administered or, alternatively, amorphogen-stimulating agent may be administered.

EXAMPLE 5 Morphogen-Induced Redifferentiation of Transformed Cells

The morphogens described herein also induce redifferentiation oftransformed cells to a morphology characteristic of untransformed cells.In particular, the morphogens are capable of inducing redifferentiationof transformed cells of neuronal origin to a morphology characteristicof untransformed neurons. The example provided below details morphogeninduced redifferentiation of a transformed cell line of neuronal origin,NG105-115. Morphogen-induced redifferentiation of transformed cells alsohas been shown in mouse neuroblastoma cells (N1E-115) and in humanembryo carcinoma cells (see copending U.S. Ser. No. 752,764,incorporated herein by reference.)

NG108-15 is a transformed hybrid cell line produced by fusingneuroblastoma×glioma cells (obtained from America Type Tissue Culture,Rockville, Md.), and exhibiting a morphology characteristic oftransformed embryonic neurons, e.g., having a fibroblastic morphology.Specifically, the cells have polygonal cell bodies, short, spike-likeprocesses and make few contacts with neighboring cells (see FIG. 2A).Incubation of NG108-15 cells, cultured in a chemically defined,serum-free medium, with 0.1 to 300 ng/ml of OP-1 for four hours inducesan orderly, dose-dependent change in cell morphology.

In the experiment NG108-15 cells were subcultured on poly-L-lysinecoated 6-well plates. Each well contained 40-50,000 cells in 2.5 ml ofchemically defined medium. On the third day 2.5 ml of OP-1 in 60%ethanol containing 0.025% trifluoroacetic was added to each well. OP-1concentrations of 0-300 ng/ml were tested. Typically, the media waschanged daily with new aliquots of OP-1, although morphogenesis can beinduced by a single four hour incubation with OP-1. In addition, OP-1concentrations of 10 ng/ml were sufficient to induce redifferentiation.After one day, hOP-1-treated cells undergo a significant change in theircellular ultrastructure, including rounding of the soma, increase inphase brightness and extension of the short neurite processes. After twodays, cells treated with OP-1 begin to form epithelioid sheets, whichprovide the basis for the growth of mutilayered aggregates at threeday's post-treatment. By four days, the great majority of OP-1-treatedcells are associated in tightly-packed, mutilayered aggregates (FIG.2B). FIG. 4 plots the mean number of multi-layered aggregates or cellclumps identified in twenty randomly selected fields from sixindependent experiments, as a function of morphogen concentration. Fortyng/ml of OP-1 is sufficient for half maximum induction of cellaggregation.

The morphogen-induced redifferentiation occurred without any associatedchanges in DNA synthesis, cell division, or cell viability, making itunlikely that the morphologic changes were secondary to celldifferentiation or a toxic effect of hOP-1. Moreover, the OP-1-inducedmorphogenesis does not inhibit cell division, as determined by³H-thymidine uptake, unlike other molecules which have been shown tostimulate differentiation of transformed cells, such as butyrate, DMSO,retinoic acid or Forskolin. The data indicate that OP-1 can maintaincell stability and viability after inducing redifferentiation. Inaddition, the effects are morphogen specific, and redifferentiation isnot induced when NG108-15 cells are incubated with 0.1-40 ng/ml TGF-β.

The experiments also have been performed with highly purified solublemorphogen (e.g., mature OP-1 associated with its pro domain) which alsospecifically induced redifferentiation of NG108-15 cells.

The morphogens described herein accordingly provide useful therapeuticagents for the treatment of neoplasias and neoplastic lesions of thenervous system, particularly in the treatment of neuroblastomas,including retinoblastomas, and gliomas. The morphogens themselves may beadministered or, alternatively, a morphogen-stimulating agent may beadministered.

EXAMPLE 6 Nerve Tissue Protection from Chemical Trauma

The ability of the morphogens described herein to enhance survival ofneuronal cells and to induce cell aggregation and cell-cell adhesion inredifferentiated cells, indicates that the morphogens will be useful astherapeutic agents to maintain neural pathways by protecting the cellsdefining the pathway from the damage caused by chemical trauma. Inparticular, the morphogens can protect neurons, including developingneurons, from the effects of toxins known to inhibit the proliferationand migration of neurons and to interfere with cell-cell adhesion.Examples of such toxins include ethanol, one or more of the toxinspresent in cigarette smoke, and a variety of opiates. The toxic effectsof ethanol on developing neurons induces the neurological damagemanifested in fetal alcohol syndrome. The morphogens also may protectneurons from the cytotoxic effects associated with excitatory aminoacids such as glutamate.

For example, ethanol inhibits the cell-cell adhesion effects induced inmorphogen-treated NG108-15 cells when provided to these cells at aconcentration of 25-50 mM. Half maximal inhibition can be achieved with5-10 mM ethanol, the concentration of blood alcohol in an adultfollowing ingestion of a single alcoholic beverage. Ethanol likelyinterferes with the homophilic binding of CAMs between cells, ratherthan their induction, as morphogen-induced N-CAM levels are unaffectedby ethanol. Moreover, the inhibitory effect is inversely proportional tomorphogen concentration. Accordingly, it is envisioned thatadministration of a morphogen or morphogen-stimulating agent to neurons,particularly developing neurons, at risk of damage from exposure totoxins such as ethanol, may protect these cells from nerve tissue damageby overcoming the toxin's inhibitory effects. The morphogens describedherein also are useful in therapies to treat damaged neural pathwaysresulting from a neuropathy induced by exposure to these toxins.

EXAMPLE 7 Morphogen-Induced CAM Expression

The morphogens described herein induce CAM expression, particularlyN-CAM expression, as part of their induction of morphogenesis. CAMs aremorphoregulatory molecules identified in all tissues as an essentialstep in tissue development. N-CAMs, which comprise at least 3 isoforms(N-CAM-180, N-CAM-140 and N-CAM-120, where “180”, “140” and “120”indicate the apparent molecular weights of the isoforms as measured bypolyacrylamide gel electrophoresis) are expressed at least transientlyin developing tissues, and permanently in nerve tissue. Both theN-CAM-180 and N-CAM-140 isoforms are expressed in both developing andadult tissue. The N-CAM-120 isoform is found only in adult tissue.Another neural CAM is L1.

N-CAMs are implicated in appropriate neural development, includingappropriate neurulation, neuronal migration, fasciculation, andsynaptogenesis. Inhibition of N-CAM production, as by complexing themolecule with an N-CAM-specific antibody, inhibits retina organization,including retinal axon migration, and axon regeneration in theperipheral nervous system, as well as axon synapses with target musclecells. In addition, significant evidence indicates that physical orchemical trauma to neurons, oncogenic transformation and some geneticneurological disorders are accompanied by changes in CAM expression,which alter the adhesive or migratory behavior of these cells.Specifically, increased N-CAM levels are reported in Huntington'sdisease striatum (e.g., striatal basal ganglia), and decreased adhesionis noted in Alzheimer's disease.

The morphogens described herein stimulate CAM production, particularlyL1 and N-CAM production, including all three isoforms of the N-CAMmolecule. For example, N-CAM expression is stimulated significantly inmorphogen-treated NG108-15 cells. Untreated NG108-15 cells exhibit afibroblastic, or minimally differentiated morphology and express onlythe 180 and 140 isoforms of N-CAM normally associated with a developingcell. Following morphogen treatment, these cells exhibit a morphologycharacteristic of adult neurons and express enhanced levels of all threeN-CAM isoforms. Using a similar protocol as described in the examplebelow, morphogen treatment of NG108-15 cells also induced L1 expression.

In this example, NG108-15 cells were cultured for four days in thepresence of increasing concentrations of OP-1 and standard Western blotsperformed on whole cell extracts. N-CAM isoforms were detected with anantibody which cross-reacts with all three isoforms, mAb H28.123,obtained from Sigma Chemical Co., St. Louis, the different isoformsbeing distinguishable by their different mobilities on anelectrophoresis gel. Control NG108-15 cells (untreated) express both the140 kDa and the 180 kDa isoforms, but not the 120 kDa, as determined bywestern blot analyses using up to 100 mg of protein. Treatment ofNG108-15 cells with OP-1 resulted in a dose-dependent increase in theexpression of the 180 kDa and 140 kDa isoforms, as well as the inductionof the 120 kDa isoform. See FIG. 3. FIG. 3B is a Western blot ofOP-1-treated NG108-15 cell extracts, probed with mAb H28.123, showingthe induction of all three isoforms. FIG. 3A is a dose response curve ofN-CAM-180 and N-CAM-140 induction as a function of morphogenconcentration. N-CAM-120 is not shown in the graph, as it could not bequantitated in control cells. However, as is clearly evident from theWestern blot in FIG. 3B, N-CAM-120 is induced in response to morphogentreatment. The increase in N-CAM expression corresponded in adose-dependent manner with the morphogen induction of multicellularaggregates. Compare FIG. 3A and FIG. 4.

FIG. 4 graphs the mean number of multilayered aggregates (clumps)counted per 20 randomly selected, microscopic viewing fields in sixindependent experiments, versus the concentration of morphogen. Theinduction of the 120 isoform also indicates that morphogen-inducedredifferentiation of transformed cells stimulates not onlyredifferentiation of these cells from a transformed phenotype, but alsodifferentiation to a phenotype corresponding to a developed cell.Standard immunolocalization studies performed with the mAb H28.123 onmorphogen-treated cells show N-CAM cluster formation associated with theperiphery and processes of treated cells, and no reactivity withuntreated cells. Moreover, morphogen treatment does not appear toinhibit cell division as determined by cell counting or ³H-thymidineuptake. Finally, known chemical differentiating agents, such asForskolin and dimethylsulfoxide, do not induce N-CAM production.

In addition, the cell aggregation effects of OP-1 on NG108-15 cells canbe inhibited with anti-N-CAM antibodies or antisense N-CAMoligonucleotides. Antisense oligonucleotides can be madesynthetically-on a nucleotide synthesizer, using standard means known inthe art. Preferably, phosphorothioate oligonucleotides (“S-oligos”) areprepared, to enhance transport of the nucleotides across cell membranes.Concentrations of both N-CAM antibodies and N-CAM antisenseoligonucleotides sufficient to inhibit N-CAM induction also inhibitedformation of multilayered cell aggregates. Specifically, incubation ofmorphogen-treated NG108-15 cells with 0.3-3 mM N-CAM antisense S-oligos,5-500 mM unmodified N-CAM antisense oligos, or 10 mg/ml mAb H28.123significantly inhibits cell aggregation. It is likely that morphogentreatment also stimulates other CAMs, as inhibition is not complete.

Finally, the above-described experiments have also been performed withsoluble morphogen (e.g., mature OP-1 dimer, associated with its prodomain polypeptides as described in Example 1). The soluble form ofmorphogen also specifically induced CAM expression.

In addition to a transformed cell line, N-CAM expression can be assayedin a primary cell culture of neural or glial cells, following theprocedures described herein. The efficacy of the morphogens describedherein to affect N-CAM expression can be assessed in vitro using asuitable cell line, such as NG108-15 and the methods described herein.

As described above, preferred morphogens, inducers, or agonists of thepresent invention can induce both N-CAM expression in vitro andendochondral bone formation when implanted in vivo in a mammal inconjunction with a matrix permissive of bone morphogenesis. Thus, themethods described herein can be used to assess novel candidatemorphogens, inducers, or agonists.

The experiments also have been performed with soluble morphogen (e.g.,mature OP-1 associated with its pro domain) which also specificallyinduced CAM expression.

The morphogens described herein are useful as therapeutic agents totreat neurological disorders associated with altered CAM levels,particularly N-CAM levels, such as Huntington's chorea and Alzheimer's'disease, and the like. In clinical applications, the morphogensthemselves may be administered or, alternatively, amorphogen-stimulating agent may be administered.

The efficacy of the morphogens described herein to affect N-CAMexpression may be assessed in vitro using a suitable cell line and themethods described herein. In addition to a transformed cell line, N-CAMexpression can be assayed in a primary cell culture of neural or glialcells, following the procedures described herein. The efficacy ofmorphogen treatment on N-CAM expression in vivo may be evaluated bytissue biopsy as described in Example 10, below, and detecting N-CAMmolecules with an N-CAM-specific antibody, such as mAb H28.123, or usingthe animal model described in Example 12.

Alternatively, the level of N-CAM proteins or protein fragments presentin cerebrospinal fluid or serum also may be detected to evaluate theeffect of morphogen treatment. N-CAM molecules are known to slough offcell surfaces and have been detected in both serum and cerebrospinalfluid. In addition, altered levels of the soluble form of N-CAM areassociated with normal pressure hydrocephalus and type II schizophrenia.N-CAM fluid levels may be detected following the procedure described inExample 10 and using an N-CAM specific antibody, such as mAb H28.123.

EXAMPLE 8 Morphogen-Induced Nerve Gap Repair (PNS)

The morphogens described herein also stimulate peripheral nervous systemaxonal growth over extended distances allowing repair and regenerationof damaged neural pathways. While neurons of the peripheral nervoussystem can sprout new processes following injury, without guidance thesesproutings typically fail to connect appropriately and die. Where thebreak is extensive, e.g., greater than 5 or 10 mm, regeneration is pooror nonexistent.

In this example morphogen stimulation of nerve regeneration was assessedusing the rat sciatic nerve model. The rat sciatic nerve can regeneratespontaneously across a 5 mm gap, and occasionally across a 10 mm gap,provided that the severed ends are inserted in a saline-filled nerveguidance channel. In this experiment, nerve regeneration across a 12 mmgap was tested.

Adult female Sprague-Dawley rats (Charles River Laboratories, Inc.)weighing 230-250 g were anesthetized with intraperitoneal injections ofsodium pentobarbital 35 mg/kg body weight). A skin incision was madeparallel and just posterior to the femur. The avascular intermuscularplane between vastus lateralis and hamstring muscles were entered andfollowed to the loose fibroareolar tissue surrounding the sciatic nerve.The loose tissue was divided longitudinally thereby freeing the sciaticnerve over its full extent without devascularizing any portion. Under asurgical microscope the sciatic nerves were transected withmicroscissors at mid-thigh and grafted with an OP-1 gel graft thatseparated the nerve stumps by 12 mm. The graft region was encased in asilicone tube 20 mm in length with a 1.5 mm inner diameter, the interiorof which was filled a morphogen solution. Specifically, The central 0.12mm of the tube consisted of an OP-1 gel prepared by mixing 1 to 5 mg ofsubstantially pure CHO-produced recombinant OP-1 with approximately 100ml of MATRIGEL™ (from Collaborative Research, Inc., Bedford, Mass.), anextracellular matrix extract derived from mouse sarcoma tissue, andcontaining solubilized tissue basement membrane, including laminin, typeIV collagen, heparin sulfate, proteoglycan and entactin, inphosphate-buffered saline. The OP-1-filled tube was implanted directlyinto the defect site, allowing 4 mm on each end to insert the nervestumps. Each stump was abutted against the OP-1 gel and was secured inthe silicone tube by three stitches of commercially available surgical10-0 nylon through the epineurium, the fascicle sheath.

In addition to OP-1 gel grafts, empty silicone tubes, silicone tubesfilled with gel only and “reverse” autografts, wherein 12 mm transectedsegments of the animal's sciatic nerve were rotated 180o prior tosuturing, were grafted as controls. All experiments were performed inquadruplicate. All wounds were closed by wound clips that were removedafter 10 days. All rats were grafted on both legs. At 3 weeks theanimals were sacrificed, and the grafted segments removed and frozen ondry ice immediately. Frozen sections then were cut throughout the graftsite, and examined for axonal regeneration by immunofluorescent stainingusing anti-neurofilament antibodies labeled with flurocein (obtainedfrom Sigma Chemical Co., St. Louis).

Regeneration of the sciatic nerve occurred across the entire 12 mmdistance in all graft sites wherein the gap was filled with the OP-1gel. By contrast, empty silicone tubes and reverse autografts did notshow nerve regeneration, and only one graft site containing the gelalone showed axon regeneration.

EXAMPLE 9 Morphogen-Induced Nerve Gap Repair (CNS)

Following axonal damage in vivo the CNS neurons are unable to resproutprocesses. Accordingly, trauma to CNS nerve tissue, including the spinalcord, optic nerve and retina, severely damages or destroys the neuralpathways defined by these cells. Peripheral nerve grafts have been usedin an effort to bypass CNS axonal damage. Successful autologous graftrepair to date apparently requires that the graft site occur near theCNS neuronal cell body, and a primary result of CNS axotomy is neuronalcell death. The efficacy of morphogens described-herein on CNS nerverepair, may be evaluated using a rat crushed optic nerve model such asthe one described by Bignami, et al., Exp. Eye Res. 28: 63-69 (1979),the disclosure of which is incorporated herein by reference. Briefly,and as described therein, laboratory rats (e.g., from Charles RiverLaboratories, Wilmington, Mass.) are anesthetized using standardsurgical procedures, and the optic nerve crushed by pulling the eyegently out of the orbit, inserting a watchmaker forceps behind theeyeball and squeezing the optic nerve with the forceps for 15 seconds,followed by a 30 second interval and second 15 second squeeze. Rats aresacrificed at different time intervals, e.g., at 48 hours, and at 3, 4,11, 15 and 18 days after operation. The effect of morphogen on opticnerve repair can be assessed by performing the experiment in duplicateand providing morphogen or PBS (e.g., 25 ml solution, and 25 mgmorphogen) to the optic nerve, e.g., just prior to the operation,concomitant with the operation, or at specific times after theoperation.

In the absence of therapy, the surgery induces glial scarring of thecrushed nerve, as determine d by immunofluorescence staining for glialfibrillary acidic protein (GFA), a marker protein for glial scarring,and by histology. Indirect immunofluorescence on air-dried cryostatsections as described in Bignami, et al., J. Comp. Neur. 153: 27-38(1974), using commercially available antibodies to GFA (e.g., SigmaChemical Co., St. Louis). Reduced levels of GFA are anticipated inanimals treated with the morphogen, evidencing the ability of morphogensto inhibit glial scar formation and to stimulate optic nerveregeneration.

EXAMPLE 10 Nerve Tissue Diagnostics

Morphogen localization in nerve tissue can be used as part of a methodfor diagnosing a neurological disorder or neuropathy. The method may beparticularly advantageous for diagnosing neuropathies of brain tissue.Specifically, a biopsy of brain tissue is performed on a patient atrisk, using standard procedures known in the medical art. Morphogenexpression associated with the biopsied tissue then is assessed usingstandard methodologies, as by immunolocalization, using standardimmunofluorescence techniques in concert with morphogen-specificantisera or monoclonal antibodies. Specifically, the biopsied tissue isthin sectioned using standard methodologies known in the art, andfluorescently labelled (or otherwise detectable) antibodies incubatedwith the tissue under conditions sufficient to allow specificantigen-antibody complex formation. The presence and quantity of complexformed then is detected and compared with a predetermined standard orreference value. Detection of altered levels of morphogen present in thetissue then may be used as an indicator of tissue dysfunction.Alternatively, fluctuation in morphogen levels may be assessed bymonitoring morphogen transcription levels, either by standard northernblot analysis or in situ hybridization, using a labelled probe capableof hybridizing specifically to morphogen RNA and standard RNAhybridization protocols well described in the art.

Fluctuations in morphogen levels present in the cerebrospinal fluid orbloodstream also may be used to evaluate nerve tissue viability. Forexample, morphogens are detected associated with adendema cells whichare known to secrete factors into the cerebrospinal fluid, and arelocalized generally associated with glial cells, and in theextracellular matrix, but not with neuronal cell bodies. Accordingly,the cerebrospinal fluid may be a natural means of morphogen transport.Alternatively, morphogens may be released from dying cells intocerebrospinal fluid. In addition, OP-1 recently has been identified inhuman blood, which also may be a means of morphogen transport, and/or arepository for the contents of dying cells.

Spinal fluid may be obtained from an individual by a standard lumbarpuncture, using standard methodologies known in the medical art.Similarly, serum samples may be obtained by standard venipuncture andserum prepared by centrifugation at 3,000 RPM for ten minutes. Thepresence of morphogen in the serum or cerebral spinal fluid then may beassessed by standard Western blot (immunoblot), ELISA or RIA procedures.Briefly, for example, with the ELISA, samples may be diluted in anappropriate buffer, such as phosphate-buffered saline, and 50 mlaliquots allowed to absorb to flat bottomed wells in microtitre platespre-coated with morphogen-specific antibody, and allowed to incubate for18 hours at 4° C. Plates then may be washed with a standard buffer andincubated with 50 ml aliquots of a second morphogen-specific antibodyconjugated with a detecting agent, e.g., biotin, in an appropriatebuffer, for 90 minutes at room temperature. Morphogen-antibody complexesthen may be detected using standard procedures.

Alternatively, a morphogen-specific affinity column may be createdusing, for example, morphogen-specific antibodies adsorbed to a columnmatrix, and passing the fluid sample through the matrix to selectivelyextract the morphogen of interest. The morphogen then is eluted. Asuitable elution buffer may be determined empirically by determiningappropriate binding and elution conditions first with a control (e.g.,purified, recombinantly-produced morphogen.) Fractions then are testedfor the presence of the morphogen by standard immunoblot, and confirmedby N-terminal sequencing. Morphogen concentrations in serum or otherfluid samples then may be determined using standard proteinquantification techniques, including by spectrophotometric absorbance orby quantitation by ELISA or RIA antibody assays. Using this procedure,OP-1 has been identified in serum.

OP-1 was detected in human serum using the following assay. A monoclonalantibody raised against mammalian, recombinantly produced OP-1 usingstandard immunology techniques well described in the art and describedgenerally in Example 14, was immobilized by passing the antibody over anactivated agarose gel (e.g., Affi-Gel™, from Bio-Rad Laboratories,Richmond, Calif., prepared following manufacturer's instructions), andused to purify OP-1 from serum. Human serum then was passed over thecolumn and eluted with 3M K-thiocyanate. K-thiocyanante fractions thenwere dialyzed in 6M urea, 20 mM PO₄, pH 7.0, applied to a C8 HPLCcolumn, and eluted with a 20 minute, 25-50% acetonitrile/0.1% TFAgradient. Mature, recombinantly produced OP-1 homodimers elute between20-22 minutes. Fractions then were collected and tested for the presenceof OP-1 by standard immunoblot. FIG. 5 is an immunoblot showing OP-1 inhuman sera under reducing and oxidized conditions. In the figure, lanes1 and 4 are OP-1 standards, run under oxidized (lane 1) and reduced(lane 4) conditions. Lane 5 shows molecular weight markers at 17, 27 and39 kDa. Lanes 2 and 3 are human sera OP-1, run under oxidized (lane 2)and reduced (lane 3) conditions.

Morphogens may be used in diagnostic applications by comparing thequantity of morphogen present in a body fluid sample with apredetermined reference value, with fluctuations in fluid morphogenlevels indicating a change in the status of nerve tissue. Alternatively,fluctuations in the level of endogenous morphogen antibodies may bedetected by this method, most likely in serum, using an antibody orother binding protein capable of interacting specifically with theendogenous morphogen antibody. Detected fluctuations in the levels ofthe endogenous antibody may be used as indicators of a change in tissuestatus.

EXAMPLE 11 Alleviation of Immune Response-Mediated Nerve Tissue Damage

The morphogens described herein may be used to alleviateimmunologically-related damage to nerve tissue. Details of this damageand the use of morphogens to alleviate this-injury are disclosed incopending U.S. Ser. No. 753,059, filed Aug. 30, 1991, the disclosure ofwhich is incorporated herein. A primary source of such damage to nervetissue follows hypoxia or ischemia-reperfusion of a blood supply to aneural pathway, such as may result from an embolic stroke, or may beinduced during a surgical procedure. As described in U.S. Ser. No.753,059, morphogens have been shown to alleviate damage to myocardialtissue following ischemia-reperfusion of the blood supply to the tissue.The effect of morphogens on alleviating immunologically-related damageto nerve tissue may be assessed using methodologies and models known tothose skilled in the art and described below.

For example, the rabbit embolic stroke model provides a useful methodfor assessing the effect of morphogens on tissue injury followingcerebral ischemia-reperfusion. The protocol disclosed below isessentially that of Phillips, et al., Annals of Neurology 25: 281-285(1989), the disclosure of which is herein incorporated by reference.Briefly, white New England rabbits (2-3 kg) are anesthetized and placedon a respirator. The intracranial circulation then is selectivelycatheterized by the Seldinger technique. Baseline cerebral angiographythen is performed, employing a digital substration unit. The distalinternal carotid artery or its branches then is selectively embolizedwith 0.035 ml of 18-hour-aged autologous thrombus. Arterial occlusion isdocumented by repeat angiography immediately after embolization. After atime sufficient to induce cerebral infarcts (15 minutes or 90 minutes),reperfusion is induced by administering a bolus of a reperfusion agentsuch as the TPA analogue FB-FB-CF (e.g., 0.8 mg/kg over 2 minutes).

The effect of morphogen on cerebral infarcts can be assessed byadministering varying concentrations of morphogens, e.g., OP-1, atdifferent times following embolization and/or reperfusion. The rabbitsare sacrificed 3-14 days post embolization and their brains prepared forneuropathological examination by fixing by immersion in 10% neutralbuffered formation for at least 2 weeks. The brains then are sectionedin a coronal plane at 2-3 mm intervals, numbered and submitted forstandard histological processing in paraffin, and the degree of nervetissue necrosis determined visually. Morphogen-treated animals areanticipated to reduce or significantly inhibit nerve tissue necrosisfollowing cerebral ischemia-reperfusion in the test animals asdetermined by histology comparison with non-treated animals.

EXAMPLE 12 Animal Model for Assessing Morphogen Efficacy In Vivo

The in vivo activities of the morphogens described herein also areassessed readily in an animal model as described herein. A suitableanimal, preferably exhibiting nerve tissue damage, for example,genetically or environmentally induced, is injected intracerebrally withan effective amount of a morphogen in a suitable therapeuticformulation, such as phosphate-buffered saline, pH 7. The morphogenpreferably is injected within the area of the affected neurons. Theaffected tissue is excised at a subsequent time point and the tissueevaluated morphologically and/or by evaluation of an appropriatebiochemical marker (e.g., by morphogen or N-CAM localization; or bymeasuring the dose-dependent effect on a biochemical marker for CNSneurotrophic activity or for CNS tissue damage, using for example, glialfibrillary acidic protein as the marker. The dosage and incubation timewill vary with the animal to be tested. Suitable dosage ranges fordifferent species may be determined by comparison with establishedanimal models. Presented below is an exemplary protocol for a rat brainstab model.

Briefly, male Long Evans rats, obtained from standard commercialsources, are anesthetized and the head area prepared for surgery. Thecalvariae is exposed using standard surgical procedures and a holedrilled toward the center of each lobe using a 0.035K wire, justpiercing the calvariae. 25 ml solutions containing either morphogen(e.g., OP-1, 25 mg) or PBS then is provided to each of the holes byHamilton syringe. Solutions are delivered to a depth approximately 3 mmbelow the surface, into the underlying cortex, corpus callosum andhippocampus. The skin then is sutured and the animal allowed to recover.

Three days post surgery, rats are sacrificed by decapitation and theirbrains processed for sectioning. Scar tissue formation is evaluated byimmunofluorescence staining for glial fibrillary acidic protein, amarker protein for glial scarring, to qualitatively determine the degreeof scar formation. Glial fibrillary acidic protein antibodies areavailable commercially, e.g., from Sigma Chemical Co., St. Louis, Mo.Sections also are probed with anti-OP-1 antibodies to determine thepresence of OP-1. Reduced levels of glial fibrillary acidic protein areanticipated in the tissue sections of animals treated with themorphogen, evidencing the ability of morphogens to inhibit glial scarformation and stimulate nerve regeneration.

EXAMPLE 13 In Vitro Model for Evaluating Morphogen Species TransportAcross the Blood-Brain Barrier

Described below is an in vitro method for evaluating the facility withwhich selected morphogen species likely will pass across the blood-brainbarrier. A detailed description of the model and protocol are providedby Audus, et al., Ann. N. Acad. Sci. 507: 9-18 (1987), the disclosure ofwhich is incorporated herein by reference.

Briefly, microvessel endothelial cells are isolated from the cerebralgray matter of fresh bovine brains. Brains are obtained from a localslaughter house and transported to the laboratory in ice cold minimumessential medium (MEM) with antibiotics. Under sterile conditions thelarge surface blood vessels and meninges are removed using standarddissection procedures. The cortical gray matter is removed byaspiration, then minced into cubes of about 1 mm. The minced gray matterthen is incubated with 0.5% dispase (BMB, Indianapolis, Ind.) for 3hours at 37° C. in a shaking water bath. Following the 3 hour digestion,the mixture is concentrated by centrifugation (1000×g for 10 min.), thenresuspended in 13% dextran and centrifuged for 10 min. at 5800×g.Supernatant fat, cell debris and myelin are discarded and the crudemicrovessel pellet resuspended in 1 mg/ml collagenase/dispase andincubated in a shaking water bath for 5 hours at 37° C. After the 5-hourdigestion, the microvessel suspension is applied to a pre-established50% Percoll gradient and centrifuged for 10 min at 1000×g. The bandcontaining purified endothelial cells (second band from the top of thegradient) is removed and washed two times with culture medium (e.g., 50%MEM/50% F-12 nutrient mix). The cells are frozen (−80° C.) in mediumcontaining 20% DMSO and 10% horse serum for later use.

After isolation, approximately 5×10⁵ cells/cm² are plated on culturedishes or 5-12 mm pore size polycarbonate filters that are coated withrat collagen and fibronectin. 10-12 days after seeding the cells, cellmonolayers are inspected for confluency by microscopy.

Characterization of the morphological, histochemical and biochemicalproperties of these cells has shown that these cells possess many of thesalient features of the blood-brain barrier. These features include:tight intercellular junctions, lack of membrane fenestrations, lowlevels of pinocytotic activity, and the presence of gamma-glutamyltranspeptidase, alkaline phosphatase, and Factor VIII antigenactivities.

The cultured cells can be used in a wide variety of experiments where amodel for polarized binding or transport is required. By plating thecells in multi-well plates, receptor and non-receptor binding of bothlarge and small molecules can be conducted. In order to conducttransendothelial cell flux measurements, the cells are grown on porouspolycarbonate membrane filters (e.g., from Nucleopore, Pleasanton,Calif.). Large pore size filters (5-12 mm) are used to avoid thepossibility of the filter becoming the rate-limiting barrier tomolecular flux. The use of these large-pore filters does not permit cellgrowth under the filter and allows visual inspection of the cellmonolayer.

Once the cells reach confluency, they are placed in a side-by-sidediffusion cell apparatus (e.g., from Crown Glass, Sommerville, N.J.).For flux measurements, the donor chamber of the diffusion cell is pulsedwith a test substance, then at various times following the pulse, analiquot is removed from the receiver chamber for analysis. Radioactiveor fluorescently-labelled substances permit reliable quantitation ofmolecular flux. Monolayer integrity is simultaneously measured by theaddition of a non-transportable test substance such as sucrose or inulinand replicates of at least 4 determinations are measured in order toensure statistical significance.

EXAMPLE 14 Screening Assay for Candidate. Compounds which AlterEndogenous Morphogen Levels

Candidate compound(s) which may be administered to affect the level of agiven morphogen may be found using the following screening assay, inwhich the level of morphogen production by a cell type which producesmeasurable levels of the morphogen is determined with and withoutincubating the cell in culture with the compound, in order to assess theeffects of the compound on the cell. This can be accomplished bydetection of the morphogen either at the protein or RNA level. A moredetailed description also may be found in U.S. Ser. No. 752,861,incorporated hereinabove by reference.

14.1 Growth of Cells in Culture

Cell cultures of kidney, adrenals, urinary bladder, brain, or otherorgans, may be prepared as described widely in the literature. Forexample, kidneys may be explanted from neonatal or new born or young oradult rodents (mouse or rat) and used in organ culture as whole orsliced (1-4 mm) tissues. Primary tissue cultures and established celllines, also derived from kidney, adrenals, urinary, bladder, brain,mammary, or other tissues may be established in multiwell plates (6 wellor 24 well) according to conventional cell culture techniques, and arecultured in the absence or presence of serum for a period of time (1-7days). Cells may be cultured, for example, in Dulbecco's Modified Eaglemedium (Gibco, Long Island, N.Y.) containing serum (e.g., fetal calfserum at 1%-10%, Gibco) or in serum-deprived medium, as desired, or indefined medium (e.g., containing insulin, transferrin, glucose, albumin,or other growth factors).

Samples for testing the level of morphogen production includes culturesupernatants or cell lysates, collected periodically and evaluated forOP-1 production by immunoblot analysis (Sambrook et al., eds., 1989,Molecular Cloning, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.),or a portion of the cell culture itself, collected periodically and usedto prepare polyA+ RNA for RNA analysis. To monitor de novo OP-1synthesis, some cultures are labeled according to conventionalprocedures with an ³⁵S-methionine/³⁵S-cysteine mixture for 6-24 hoursand then evaluated to OP-1 synthesis by conventional immunoprecipitationmethods.

14.2 Determination of Level of Morphogenic Protein

In order to quantitate the production of a morphogenic protein by a celltype, an immunoassay may be performed to detect the morphogen using apolyclonal or monoclonal antibody specific for that protein. Forexample, OP-1 may be detected using a polyclonal-antibody specific forOP-1 in an ELISA, as follows.

1 mg/100 ml of affinity-purified polyclonal rabbit IgG specific for OP-1is added to each well of a 96-well plate and incubated at 37° C. for anhour. The wells are washed four times with 0.167M sodium borate bufferwith 0.15 M NaCl (BSB), pH 8.2, containing 0.1% Tween 20. To minimizenon-specific binding, the wells are blocked by filling completely with1% bovine serum albumin (BSA) in BSB and incubating for 1 hour at 37° C.The wells are then washed four times with BSB containing 0.1% Tween 20.A 100 ml aliquot of an appropriate dilution of each of the test samplesof cell culture supernatant is added to each well in triplicate andincubated at 37° C. for 30 min. After incubation, 100 ml biotinylatedrabbit anti-OP-1 serum (stock solution is about 1 mg/ml and diluted1:400 in BSB containing 1% BSA before use) is added to each well andincubated at 37° C. for 30 min. The wells are then washed four timeswith BSB containing 0.1% Tween 20. 100 ml strepavidin-alkaline (SouthernBiotechnology Associates, Inc. Birmingham, Ala., diluted 1:2000 in BSBcontaining 0.1% Tween 20 before use) is added to each well and incubatedat 37° C. for 30 min. The plates are washed four times with 0.5M Trisbuffered Saline (TBS), pH 7.2. 50 ml substrate (ELISA AmplificationSystem Kit, Life Technologies, Inc., Bethesda, Md.) is added to eachwell incubated at room temperature for 15 min. Then, 50 ml amplifier(from the same amplification system kit) is added and incubated foranother 15 min at room temperature. The reaction is stopped by theaddition of 50 ml 0.3 M sulfuric acid. The OD at 490 nm of the solutionin each well is recorded. To quantitate OP-1 in culture media, a OP-1standard curve is performed in parallel with the test samples.

Polyclonal antibody may be prepared as follows. Each rabbit is given aprimary immunization of 100 μg/500 ml E. coli produced OP-1 monomer(amino acids 328-431 in SEQ ID NO:5) in 0.1% SDS mixed with 500 μl E.coli produced OP-1 monomer (amino acids 328-431 in SEQ ID NO: 5) in 0.1%SDS mixed with 50011 Complete Freund's Adjuvant. The antigen is injectedsubcutaneously at multiple sites on the back and flanks of the animal.The rabbit is boosted after a month in the same manner using incompleteFreund's Adjuvant. Test bleeds are taken from the ear vein seven dayslater. Two additional boosts and test bleeds are performed at monthlyintervals until antibody against OP-1 is detected in the serum using anELISA assay. Then, the rabbit is boosted monthly with 100 mg of antigenand bled (15 ml per bleed) at days seven and ten after boosting.

Monoclonal antibody specific for a given morphogen may be prepared asfollows. A mouse is given two injections of E. coli produced OP-1monomer. The first injection contains 100 mg of OP-1 in completeFreund's adjuvant and is given subcutaneously. The second injectioncontains 50 mg of OP-1 in incomplete adjuvant and is givenintraperitoneally. The mouse then receives a total of 230 mg of OP-1(amino acids. 307-431 in SEQ ID NO:5) in four intraperitoneal injectionsat various times over an eight month period. One week prior to fusion,both mice are boosted intraperitoneally with 100 mg of OP-1 (307-431)and 30 mg of the N-terminal peptide (Ser₂₉₃-Asn₃₀₉-Cys) conjugatedthrough the added cysteine to bovine serum albumin with SMCCcrosslinking agent. This boost was repeated five days (IP), four days(IP), three days (IP) and one day (IV) prior to fusion. The mouse spleencells are then fused to myeloma (e.g., 653) cells at a ratio of 1:1using PEG 1500 (Boeringer Mannheim), and the cell fusion is plated andscreened for OP-1-specific antibodies using OP-1 (307-431) as antigen.The cell fusion and monoclonal screening then are according to standardprocedures well described in standard texts widely available in the art.

EXAMPLE 15 Morphogen-Induced Dendritic Growth in Spinal Motor Neurons InVitro

In order to evaluate the effects of various neurotrophic proteins onneurite outgrowth, dissociated motor neurons from the spinal cord wereexposed OP-1, BDNF, LIF, or GDNF in vitro.

Suspensions of motor neurons dissociated from the spinal cord of ratfetuses (E14 day) were prepared and plated essentially according to themethod of Higgins, et al., CULTURING NERVE CELLS, Banker and Goslin,eds., MIT Press, pp. 177-205 (1991), incorporated by reference herein.Neurons were plated at low density (about 15 cells/mm²) ontopoly-D-lysine coated coverslips and maintained in a serum-free medium,Higgins, et al., CULTURING NERVE CELLS (1991). Cytosine-b-D-furanoside(1 μM) was added to the medium of all cultures for 48 hrs on the secondday. This exposure was sufficient to render the cultures virtually freeof normeuronal cells for 30 days. Exposure to vehicle, OP-1, BDNF, LIF,or GDNF was initiated after the elimination of normeuronal cells.

Cellular morphology was routinely assessed by intracellular injection offluorescent dyes (4% Lucifer Yellow or 5% 5,6 dicarboxyfluorescein;Bruckenstein and Higgins, Dev. Biol. 128: 924-936 (1988). Only neuronswhose cell bodies were at least 150 mm from their nearest neighbor wereinjected, because density-dependent changes in morphology occur whensomata of motor neurons are separated by lesser distances. Highlypurified recombinant human OP-1 was isolated from medium conditioned bytransfected Chinese hamster ovary cells using S-Sepharose andphenyl-Sepharose chromatography followed by reverse phase highperformance liquid chromatography as described previously. See Sampath,et al., J. Biol. Chem. 267: 20352-20362 (1992).

Cultures were immunostained with antibodies previously shown to reactselectively with either axons or dendrites. Lein and Higgins, Dev. Biol.136: 330-345 (1989). Dendritic probes included mAb to MAP2, tononphosphorylated forms of the M and H neurofilaments, and to thetransferrin receptor. Axonal probes included monoclonal antibodiesagainst to synaptophysin, tau, and phosphorylated forms of the H and theM and H neurofilament subunits. All antigens were localized by indirectimmunofluorescence using previously described procedures. Lein andHiggins, Dev. Biol. 136: 330-345 (1989). Image 1 Software (UniversalImaging) was used for the morphometric analyses of dendritic growth inimmunostained cultures.

Addition of OP-1 enhanced dendritic growth of spinal motor neurons. Thedendrites that formed in the presence of OP-1 were significantly longer,had a larger number of branchpoints, and had a larger diameter thancontrol spinal motor neurons. The effects of OP-1 appeared to bespecific to dendrites OP-1 did not significantly affect the total lengthof axons. However, axon length was significantly increased by LIF andGDNF. These observations are summarized in Table I. TABLE I COMPARISONOF THE EFFECTS OF VARIOUS GROWTH HORMONES ON DENDRITIC GROWTH OF SPINALMOTOR NEURONS Total dendritic # of dendritic Condition lengthbranchpoints Least somal diameter Total axonal length Control 65.09 ±6.7  0.65 ± 0.17 8.81 ± 0.32 180.95 ± 14.5  OP-1 133.38 ± 10.0*   1.40 ±0.22* 10.10 ± 0.44* 200.20 ± 11.9  BDNF 56.58 ± 8.6  0.45 ± 0.78 9.56 ±0.32 158.10 ± 9.8   LIF 59.64 ± 8.3  0.58 ± 0.20 8.87 ± 0.29 272.00 ±24.2*  GDNF 52.06 ± 12.6  0.76 ± 0.39 8.66 ± 0.24 288.89 ± 23.5* All measurements are in micrometers. Data are expressed as the mean ±SEM.N = 40.*Significantly different from Control group.

EXAMPLE 16 Morphogen-Induced Dendritic Growth in Various Neurons InVitro

In order to further evaluate the effects of morphogens on neuriteoutgrowth, various neuronal populations were exposed OP-1 in vitro.

16.1 Cortical Neurons

The effects of morphogens on neurite outgrowth were evaluated incortical neurons. Pregnant Balb/c mice (E8) were euthanised bydecapitation following CO₂ anesthesia and the embryos removed understerile conditions. After carefully removing the meninges, the frontalcerebral cortex was dissected in sterile Hank's balanced salt solution(HBSS) without Ca⁺²/Mg⁺² (Biowhittaker) containing 0.6% glucose and 0.5%HEPES (Sigma). The cortex was minced to 1 mm thick pieces anddissociated into a single-cell suspension using the following protocol.Pieces of frontal cortex were placed in 4.5 ml of Ca⁺²/Mg⁺²-free HBSS ina 50 ml conical culture tube and incubated in a water bath for 5 minutesat 37° C. Then, 0.5 ml of 2.5% trypsin solution (Gibco) was added andthe tissue was then incubated for 10 minutes on a shaking device at 37°C. The supernatant was then removed and placed into another tubecontaining 0.5 ml fetal bovine serum (FBS; Gibco). Five ml of 0.025%Deoxyribonuclease I (Dnase; Calbiochem Corp.) in Ca⁺²/Mg⁺²-free HBSS wasthen added to the pellet and the incubation was continued for another 5min on a shaking device at 37° C. At the end of incubation, the trypsinwas inactivated by adding 0.5 ml FBS. The supernatant collected earlierwas combined with the tissue and the cells were then concentrated bycentrifugation (1000 rpm, 5 min.) and the supernatant was decanted.Fresh medium (2 ml) was added to resuspend the pellet which was furtherdissociated into a single-cell suspension by trituration using apipet-tip.

The cells were plated in Neurobasal Medium (GIBCO), and supplementedwith B-27 Supplement (GIBCO), 1 mM glutamine andpenicillin/streptomycin. For all experiments, the cortical neurons wereplated at low density (1×10⁴ cells/0.5 ml) on poly-D-lysine (50-100μg/ml; Sigma) coated coverslips inserted into 24-well culture plate(Falcon). Cells were grown for two days in vitro at 37° C. in anatmosphere of 5% CO₂. OP-1 (1, 10, 30, or 100 ng/ml) was added eitherthree hours or 24 hours after plating the cortical neurons. BSA wasadded to all wells at a final concentration of 500 μg/ml prior to addingOP-1. Control cultures consisted of culture medium and medium with BSA500 μg/ml.

Mouse neurites were immunostained with M6, a mouse neuron-specificmonoclonal antibody. Cells were first incubated in 0.1 M phosphatebuffered saline (PBS) containing 1% BSA for 30 min at 25° C. and thenexposed to M6 in PBS (1:10) for 24 hrs at 4° C. Immunofluorescentlabeling for M6 was carried out using biotinylated secondary antibodiesanti-rat IgG (Sigma; 3 μg/ml, 1:200, 1 hr at 37° C.) followed byavidin-TRITC conjugate (Sigma; 6.5 μg/ml, 1:400, 1 hr at 37° C., in thedark).

Axons were identified with a rabbit polyclonal antiserum to the 200 kDaneurofilament protein (NF-H; Sigma; 1:100, 91 μg/ml). A monoclonalantibody to microtubule-associated protein 2 (MAP2; Boehringer-Mannheim;1:100, 20 μg/ml) was used as a specific marker of dendrites. Tovisualize these intracellular antigens, cells were permeabilized with0.5% triton X-100 (TX) in 0.1 M PBS containing 1% BSA and 4% goat serum(GS; Sigma) for 1 hr at 25° C. Primary antibodies were diluted in 0.1MPBS, 1% BSA, 4% GS with 0.5% TX and incubated for 1 hr at 37° C. Afterthe primary incubation, the cells were washed three times in PBScontaining 4% GS. Labeling was detected with fluorescein- orrhodamine-conjugated antibodies (1:400 in PBS, BSA, GS, and TX, 1 hr at37° C., in the dark). Mouse antibodies were visualized withfluorescein-coupled goat anti-mouse Ig (Boehringer-Mannheim). Rabbit orrat antibodies were visualized using indirect immunofluorescence withrhodamine-conjugated goat anti-rat or anti-rabbit Ig. Cultures wereadditionally stained with a nuclear stain, 4′,6-Diamidino-2-phenylindoledihydrochloride hydrate (DAPI; Sigma; 0.1 μg/ml, 5 min at roomtemperature). Coverslips were washed once in sterile water and let dryfor 10 minutes before mounting onto glass slides in aqueous mountingsolution (Fluoromount; Southern Biotechnology). Slides were keptrefrigerated in the dark until examined.

Immunoreactive cells were examined in six different microscopic fieldsselected at random on a minimum of five coverslips for each experiment.Three experiments for each condition were carried out. Only isolatedneurons whose cell bodies or processes that were not in contact withother neurons were analyzed. A total of 100 neurons were examined foreach experimental condition.

For measurements of neurite length, neurons were examined at a finalimage magnification of 400×. Fluorescent images of the neurons wererecorded with a CCD video camera (Dage) and analyzed with a MacintoshPowerMac (9500/200) and image processing program (NIH Image 1.59).Neurite lengths were measured by tracing the total length of any neuriteextending from a neuron cell body. Recorded lengths were calibrated atthe same magnification using a ciroscope slide micrometer. The number ofprimary dendrites per cell, the length of major neurites or axons, thelength of primary dendrites, the length and number of secondarydendrites, the total length of primary or secondary dendrites, and thetotal neurite and dendrite length per cell were calculated. Analysis ofstatistical significance of any observed differences between monolayerswas performed using Student's t-test or ANOVA (SPSS/Mac, version 6.1,SPSS Inc., Chicago, Ill.).

OP-1 enhanced dendritic growth of cortical neurons. The length ofprimary dendrites and the number and length of secondary dendrites weresignificantly increased in the presence of OP-1. The increase indendritic growth was dose-dependent. Maximal growth was observed in thepresence of 30-100 μg/ml of OP-1. The effects of OP-1 appeared to bedendrite-specific; OP-1 did not significantly affect the cellmorphology, body size, and axon length of cortical neurons. Theseobservations are summarized in Table II. TABLE II EFFECTS OF OP-1 ONDENDRITIC GROWTH OF CORTICAL NEURONS Cell Body Condition Diameter AxonLength # Primary # Secondary BSA 3 hrs 15.9 ± 0.5  153.2 ± 9.1  3.5 ±0.2  0.4 ± 0.14 OP-1, 1 μg, 3 hrs 15.98 ± 0.5  151.1 ± 6.2  3.5 ± 0.20.55 ± 0.13 OP-1, 10 μg, 3 hrs 17.8 ± 0.49 139.3 ± 8.5   4.0 ± 0.14  0.6± 0.13 OP-1, 30 μg, 3 hrs 17.74 ± 0.5  134.5 ± 6.9   4.0 ± 0.15 0.96 ±0.14 OP-1, 100 μg, 3 hrs 17.3 ± 0.4  138.8 ± 6.6  3.6 ± 0.2 1.3 ± 0.2BSA 24 hrs 16.3 ± 0.45 144.8 ± 5.9   3.5 ± 0.17 0.48 ± 0.11 Op-1, 1 μg,24 hrs 15.6 ± 0.5  142.6 ± 10.9  3.4 ± 0.2 0.4 ± 0.1 OP-1, 10 μg, 24 hrs16.7 ± 0.4  144.2 ± 8.16   3.2 ± 0.14 0.6 ± 0.1 OP-1, 30 μg, 24 hrs 16.5± 0.4  139.4 ± 7.8  3.5 ± 0.2 0.86 ± 0.12 OP-1, 100 μg, 24 hrs 17.3 ±0.4  156.5 ± 11.1   3.0 ± 0.19 0.86 ± 0.17 Length Length Total TotalTotal Condition Primary Secondary Primary Secondary Dendrite BSA 3 hrs15.4 ± 0.6  3.4 ± 0.8 51.4 ± 4.5  3.6 ± 1.5 54.9 ± 5.1  OP-1, 1 μg, 3hrs 14.2 ± 0.4  4.7 ± 0.7 49.1 ± 3.5  5.7 ± 1.5 54.8 ± 4.6  OP-1, 10 μg,3 hrs 20.5 ± 0.6  4.6 ± 0.6 81.6 ± 3.5  5.8 ± 1.2 87.5 ± 4.3  OP-1, 30μg, 3 hrs 22.9 ± 0.5  7.6 ± 0.7 89.9 ± 4.6  11.1 ± 1.9  99.6 ± 5.7 OP-1, 100 μg, 3 hrs 23.7 ± 0.6   9.9 ± 0.75 87.7 ± 5.3  16.2 ± 2.9 103.9 ± 7.6  BSA 24 hrs 16.7 ± 0.5  4.6 ± 0.6 55.9 ± 3.5  4.7 ± 1.1 60.6± 4.1  OP-1, 1 μg, 24 hrs 13.7 ± 0.5  3.7 ± 0.7 46.3 ± 3.6  4.1 ± 1.050.4 ± 4.0  OP-1, 10 μg, 24 hrs 16.3 ± 0.6  4.8 ± 0.7 50.8 ± 3.1  5.3 ±7.7 56.1 ± 3.6  OP-1, 30 μg, 24 hrs 18.9 ± 0.5  7.4 ± 0.8 65.6 ± 4.5 9.6 ± 1.6 75.5 ± 5.1  OP-1, 100 μg, 24 hrs 22.1 ± 0.8  7.6 ± 1.0 64.0 ±6.4  10.5 ± 2.2  74.8 ± 7.8 

-   -   Three separate experiments were performed for each condition. At        least 30 isolated cells, identified by MAP2 and NF-H        immunolabeling and not touching other neurons were analyzed for        each experiment after 2DIV using computer assisted image        analysis. Data are expressed as means±standard error.

16.2 Hippocampal Neurons

The effects of morphogens on neurite outgrowth were evaluated inhippocampal neurons. Primary hippocampal cultures were preparedaccording to the method of Banker, et al. See Banker & Cowan Brain Res.126: 397-425 (1977); Banker & Goslin, CULTURING NERVE CELLS (1991). Avery low density of neurons was plated over a monolayer of glial cellsplated on poly-D-lysine coated coverslips and maintained in a serum-freemedium. Cellular morphology was assessed by immunostaining for MAP2.Dendritic length and branching was quantified using the Shoil concentricring analysis. Under these control conditions, hippocampal neuronsproduce 4-6 minor processes. Over the first 24-48 hours, one of theprocesses grows rapidly and becomes an axon. The other processes extendvery slowly and develop into mature dendrites after 6-10 days.

Because glial cells secrete trophic factors into the medium that arecritical for the development of hippocampal neurons, this culture methodwas modified to assess the effects of OP-1. Hippocampal neurons wereplated in a serum-free medium without glial cells. In the absence ofglial cells, OP-1 markedly enhanced the rate and extent of dendriticdevelopment of hippocampal neurons cultured in serum-free medium.OP-1-treated neurons had significantly increased number of Shoil ringintersections (39.6 vs. 16.02), dendritic length (FIGS. 6 and 7) andnumber of terminal branches (13.05 vs. 6.64; FIG. 8). There were nosignificant differences in the number of primary dendrites. The effectsof OP-1 appeared to be dendrite-specific in this cell type. Asillustrated in FIG. 6; OP-1 did not significantly affect the totallength of axons.

16.3 Sympathetic Neurons

(i) Effects of OP-1 on Dendritic Growth

In order to assess the effects of morphogens on sympathetic neurons,suspensions of neurons dissociated from the superior cervical ganglia ofSprague-Dawley rat fetuses (19-21 day) or rat pups (1-3 day postnatal)were exposed to OP-1. The suspension were prepared according to themethod of Higgins, et al., CULTURING NERVE CELLS, Banker and Goslin,eds., MIT Press, pp. 177-205 (1991), the teachings of which hereinincorporated by reference. Equivalent results were obtained with pre-and postnatal animals. Neurons were plated at low density (about 15cells/mm²) onto poly-D-lysine coated coverslips and maintained in aserum-free medium (Higgins, et al., CULTURING NERVE CELLS (1991))containing NGF (100 ng/ml). Cytosine-b-D-furanoside (1 μM) was added tothe medium of all cultures for 48 hrs on the second day. This exposurewas sufficient to render the cultures virtually free of normeuronalcells for 30 days. To label sympathetic neuroblasts, ganglia from 15-dayembryos were grown in explant culture for 18 hrs in the presence of³H-(methyl)-thymidine (0.3 mCi/ml, ICN) before being dissociated.Because NT3 (50 ng/ml) enhances the survival of immature sympatheticneurons, Birren, et al., Develop. 119: 597-610 (1993), it was added tothe NGF-containing medium during the period of explant culture and thenext 4 days in vitro. As in cultures of sympathetic neurons, exposure toNGF, OP-1, or both, was initiated after the elimination of normeuronalcells.

Cellular morphology was routinely assessed by intracellular injection offluorescent dyes (4% Lucifer Yellow or 5% 5,6 dicarboxyfluorescein;Bruckenstein and Higgins, Dev. Biol. 128: 924-936 (1988)). Only neuronswhose cell bodies were at least 150 mm from their nearest neighbor wereinjected, because density-dependent changes in morphology occur whensomata of sympathetic neurons are separated by lesser distances. Highlypurified recombinant human OP-1 was isolated from medium conditioned bytransfected Chinese hamster ovary cells using S-Sepharose andphenyl-Sepharose chromatography followed by reverse phase highperformance liquid chromatography as described previously. See Sampath,et al., J. Biol. Chem. 267: 20352-20362 (1992).

Under control conditions, sympathetic neurons typically extended asingle process during the first 24-48 hrs in vitro. This process has thecytoskeletal and ultrastructural characteristics of an axon. The axoncontinued to elongate during the next few weeks and generate anelaborate plexus. The basic morphology of the cells, however, remainedessentially unchanged, with 80% of the neurons still being unipolarafter one month in vitro. Most of the remainder had either two axons(13% of the cells) or an axon and a short dendrite (7%). Thus, the meannumber of processes at this time was 1.13±0.06 axons/cells and 0.07±0.04dendrites/cell.

Exposure to OP-1 caused sympathetic neurons to form additionalprocesses. This response was relatively slow, with only 42% of the cellsforming a second process within 24 hrs. However, virtually all cells(94%) had begun to respond to maximally effective concentrations of OP-1within three days. The processes that formed in the presence of OP-1 hadthe appearance of dendrites. These processes were broad-based (up to 5μm diameter), exhibited a distinct taper, and branched in a “Y”-shapedpattern, with daughter processes being distinctly smaller than theparent process. Dendrites were much thicker than sympathetic axons and,unlike axons, they ended locally, usually extending less than 300 mmfrom the soma. The mean number of dendrites/cell continued to increaseduring a four week exposure to OP-1, with most of the change occurringduring the first 10 days of treatment. After four weeks, OP-1-treatedneurons had a mean of 7.3±0.3 dendrites/cell, representing a 100-foldincrease over control cells. During this time, the size of the dendriticarbor also increased, with cells progressing from simple cells to a morecomplicated morphology. These observations are summarized in panels A, Band C of FIG. 9.

The effects of OP-1 appeared to be dendrite-specific in this cell type.The effects of OP-1 on initial axon growth during the first 48 hrs inculture were examined. OP-1 did not affect the rate at which axons wereinitially extended, or the number of axons extended per cell. Cellnumber also remained constant during the exposure to OP-1, indicatingthat the morphogen was not acting by enhancing the survival of asubpopulation of neurons, as shown in panel C of FIG. 9. The effects ofOP-1 were also examined in the delayed introduction paradigm, and noincrease in axon number was observed.

(ii) Effects of Various Concentrations of OP-1 on Dendritic Growth

In order to assess whether the effects of morphogens on dendritic growthwere concentration-dependent, suspensions of neurons dissociated fromthe superior cervical ganglia were exposed to various concentrations ofOP-1.

The effects of OP-1 were concentration-dependent (FIG. 10). Significantchanges in dendritic growth could be detected with concentrations as lowas 300 pg/ml, and half-maximal effects were observed at about 2 ng/ml.Maximal dendritic growth was obtained with medial concentrations between30 and 100 ng/ml. Although typically added to the medium on day 5,earlier initiation of dendritic growth (by the third day of culture)could be obtained by adding OP-1 to the medium at the time of plating.No dendritic growth was detected in cultures in which the OP-1 (1 μg/ml)had been allowed to absorb to coverslips before plating cells.

It appeared that several parameters of dendritic growth, including thepercentage of cells with dendrites, the mean number of dendrites/cell,dendritic length (not shown), changed over the same concentration range.In addition, three other changes were observed in cellular morphology inthis morphogen concentration range. As had been observed while dendriticgrowth is occurring in vivo, the somata became larger. In addition, thenuclei became less eccentric, and the axons formed small fascicles.

(iii) Effects of Various Morphogens on Dendritic Growth

Using the methods described above, other morphogens were tested fortheir capacity to induce dendritic growth of sympathetic neurons andtheir effects on the expression of cytoskeletal proteins. Sympatheticneurons were dissociated from the superior cervical ganglia of Holtzman(Harlan Sprague-Dawley) rat fetuses (E21) or pups (1 day postnatal) andplated onto poly-D-lysine-coated coverslips according to the method ofHiggins, et al., CULTURING NERVE CELLS, Banker and Goslin, eds., MITPress, pp.177-205 (1991). The cells were maintained in a serum-freemedium that contains nerve growth factor (100 ng/ml), and normeuronalcells were eliminated by exposure to cytosine-β-D-arabinofuranoside (1μM) for 48-72 h beginning on the second day after plating. Themorphology of the neurons was routinely assessed by intracellularinjection of the fluorescent dye Lucifer yellow (4%) and byimmunostaining with dendrite-specific antibodies. These includedmonoclonal antibody SMI 32 (Stemberger Monocionals, Inc.) whichrecognizes nonphosphorylated epitopes on the H and M neurofilamentsubunits and AP20 (Sigma) and SMI 52 (Stemberger Monocionals, Inc.)which both react with microtubule-associated protein-2 (MAP2). Highlypurified recombinant human proteins (BMP-2, BMP-3 and CDMP-2) andDrosophila 60A were prepared as described previously for OP-1.

For Western blot analysis of cytoskeletal proteins, sympathetic neuronswere plated onto poly-D-lysine coated 35 mm dishes and treated with 50ng/ml of BMP-2, OP-1, 60A or CDMP-2 for five days. Cells were thenscraped off dishes in 50 mM Tris buffer (pH 7.4) containing 0.1% SDS, 2%2-mecaptoenthanol and 1 mM EDTA and homogenized by passing through a 23gauge needle at 4° C. Cell extracts were centrifuged at 12000×g for 15minutes and the protein concentrations of the supernatants weredetermined using the Bradford dye reagent (Bio-Rad). Equal amounts ofproteins were resolved by SDS-PAGE, electrophoretically transferred ontoa nitrocellulose membrane, and probed with antibodies to MAP2 or anantibody SMI 31 (Stemberger Monocionals, Inc.) to the phosphorylatedforms of the H and M neurofilament subunits. Detection was accomplishedusing Chemiluminescent Substrate (Pierce Chemical Co.) after sequentialtreatment with biotinylated goat antimouse IgG (HyClone Laboratories,Inc.) and with horseradish peroxidase-conjugated streptavidin(Amersham).

As illustrated in FIG. 11, all the morphogens tested (i.e., OP-1, BMP-2,BMP-3, CDMP-2, and 60A) induced significant dendritic growth insympathetic neurons. However, significant variations in efficacy wereobserved. Treatment with maximally effective concentrations (50 ng/ml)of BMP-2 or OP-1 for five days caused virtually all of the neurons toform dendrites (Table III). These processes exhibited a distinct taper,branched at “Y” shaped angles, and extended approximately 100 μm fromthe cell bodies after five days of treatment. Examination ofconcentration effect relationships (FIG. 1) revealed that the EC₅₀ forBMP-2 (1.7 ng/ml) was similar to that for OP-1 (1.8 ng/ml) and thatmaximally effective concentrations of these two growth factors hadequivalent effects on the cells, as assessed by both the number ofdendrites per cell and the length of the longest dendrite (Table III).Moreover, the effects of OP-1 and BMP-2 were not additive (data notshown) suggesting that the two ligands may share aspects of a commonsignaling pathway. The Drosophila 60A protein also stimulated dendriticgrowth and the EC₅₀ (2.7 ng/ml) for this activity was similar to thatfor OP-1 and BMP-2. However, 60A was less efficacious, and at maximallyeffective concentrations caused cells to form fewer dendrites (1.2/cell)than either OP-1 or BMP-2 (4.6 or 4.9/cell, respectively). BMP-3 andCDMP-2 only produced a statistically significant increase in dendriticgrowth at the highest concentration tested (100 ng/ml). The differentefficacies for promoting dendrite growth may indicate relativelystringent structural requirements for this biological activity. OP-1,BMP-2 and 60A, which share a high sequence homology (89-90%) in theconserved seven-cysteine skeleton sequence, had much higher efficaciesthan BMP-3 and CDMP-2, which share 78% and 82% homology, respectively,with the reference sequence of OP-1. See FIG. 1. TABLE III COMPARISON OFTHE EFFECTS OF VARIOUS MORPHOGENS ON DENDRITIC GROWTH OF SYMPATHETICNEURONS Length of Growth Dendrites % Cells with the Longest EC₅₀ Factorper Cell Dendrites Dendrite (pm) (ng/ml) Control 0.20 ± 0.14 13.3  6.0 ±4.3 OP-1  4.62 ± 0.49* 100.0 112.3 ± 5.6  1.84 BMP-2  4.93 ± 0.46* 100.0107.1 ± 5.6  1.66 60A  1.20 ± 0.26* 73.3 50.7 ± 9.0 2.70 BMP-3 0.44 ±0.22 25.0 16.3 ± 7.6 26.05 CDMP-2 0.25 ± 0.14 18.8 12.5 ± 6.7 98.38Sympathetic neurons were exposed to 50 ng/ml of each of the growthfactors for five days and then immunostained with a dendrite specificantibody (SMI 32). Cellular morphology was analyzed by fluorescencemicroscopy using Metamorph software Universal Imaging). Data arepresented as the mean ± SEM; N = 20-30.*p < vs control (Student's t-test).

The effects of various morphogens on the expression of cytoskeletalproteins were also assessed using methods described above. Afternormeuronal cells had been eliminated, sympathetic neurons were treatedwith control medium or with 50 ng/ml of OP-1, BMP-2, 60A or CDMP-2, forfive days. Cultures were then solubilized and subjected to Western blotanalysis for MAP2 (primarily located in dendrites) and forphosphorylated forms of the H and M neurofilament subunits (primarilylocated in axons). The efficacy of the various morphogens in increasingMAP2 expression correlated with their ability to induce dendriticgrowth. Cultures exposed to BMP-2, OP-1 or 60A exhibited significantincreases (3.0±0.4, 2.3±0.4, and 1.8±0.3 fold, respectively) in theexpression of high molecular weight forms of MAP2 when compared tocontrol cultures. The level of expression of MAP2 was not significantlyincreased in cultures exposed to CDMP-2. None of the morphogens testedaffected the expression of the phosphorylated forms of the H and Mneurofilament subunits. These results show that morphogens enhance theexpression of a microtubule-associated protein which is found indendrites and which is required for the growth of these processes. Theseobservations suggest that regulation of MAP2 expression may be one ofthe mechanisms by which morphogens regulate the morphologicaldevelopment of sympathetic neurons.

(iv) Comparison of OP-1 to Other Growth Factors

In order to evaluate whether the effects on dendritic outgrowth arespecific to morphogens, the effects of other growth factors on dendriticgrowth were compared to those of OP-1.

Mature human recombinant OP-1 was isolated from medium conditioned bytransfected Chinese hamster ovary cells using S-Sepharose andphenyl-Sepharose chromatography followed by reverse phase highperformance liquid chromatography as described above. Ciliaryneurotrophic factor (CNTF) was purified from rat sciatic nerveManthorpe, et al., Brain Res. 367: 282-286 (1986), the teachings ofwhich are herein incorporated by reference) and activin A was generouslyprovided by Ralph Schwall (Genentech). Other growth factors wereobtained from commercial sources: GIBCOBRL (IL-1β, IL-3, IL-4, IL-6,IL-7; LIF, EGF, GM-CSF, RANTES, MCAF, TGF-α, TGF-β1 and 3, rat gammainterferon); Collaborative Research (HGF, PDGF); Boehringer (IL-2); andPromega (IL-8); R&D Systems (BDNF, NT3, NT4, bFGF).

As summarized in Table IV, dendritic growth was not observed in thepresence of TGF-β1, TGF-β3, activin A or inhibin, all of which aremembers of the TGF-β family but are not members of the structurally andfunctionally distinct morphogen sub-family. In addition, negativeresults were obtained with most neurotrophins and nine other growthfactors known to affect neuronal survival or differentiation (Table IV).In other experiments, negative results were also obtained with: TGF-β2,interleukins 1β, 2, 3, 4, 6, 7, and 8, PDGF, HGF, GM-CSF, MCAF, RANTES,TGF-α and gamma interferon. Thus, it would appear that thedendrite-promoting effect of morphogens is a highly specific responsethat is observed with a very limited subset of growth factors. TABLE IVCOMPARISON OF THE EFFECTS OF OP-1 AND OTHER GROWTH FACTORS ON DENDRITICGROWTH GROWTH MEAN NUMBER OF FACTOR DENDRITES/CELL NONE  0.8 ± 0.04 OP-13.08 ± 0.20 TGF-β1 0.17 ± 0.09 TGF-β3 0.00 ± 0.00 INHIBIN 0.20 ± 0.10ACTIVIN A 0.08 ± 0.05 BDNF 0.11 ± 0.05 NT3 0.11 ± 0.07 NT4 0.32 ± 0.11CNTF 0.10 ± 0.05 LIF 0.13 ± 0.07 EGF 0.07 ± 0.07 bFGF 0.03 ± 0.03

-   -   Beginning on day 5, cultures of sympathetic neurons were exposed        to varying concentrations of growth factors. Seven to eight days        later, the mean number of dendrites/cell was assessed by        intracellular dye injection. Only the results obtained with the        highest concentration tested (100 ng/ml) are shown in this        table, but lower concentrations yielded similar results. N>30        cells for each condition

(v) Role of Morphogens in Glial-Induced Dendritic Growth

Sympathetic neurons extend only a single axon when grown in the absenceof serum or normeuronal cells. In contrast, co-culturing sympatheticneurons with glial cells causes these neurons to form dendrites. Inorder to assess the potential role of morphogens in glial-induceddendritic growth, neuron and glial cells were co-cultured in thepresence of a monoclonal antibody (mAb) raised against hOP-1.

Dendritic growth in sympathetic neurons grown with astrocytes or Schwanncells was inhibited by 40-60% in the presence of a hOP-1 mAb. SDS-PAGEanalyses by hOP-1 mAb of proteins immunoprecipitated from neuron-gliaco-cultures revealed several bands, the molecular weights of whichcorresponded to the cellular and secreted forms of hOP-1.Immunocytochemical analyses of co-cultures indicate that both neuronsand glia express cytoplasmic and surface staining for OP-1 and BMP-6.Similar patterns of immunoreactivity were observed in glia grown in theabsence of neurons. However, neurons cultured in the absence of gliaexpressed cytoplasmic but not surface staining for OP-1 or BMP-6. Thesedata are consistent with a role for morphogens in glia-induced dendriticgrowth.

EXAMPLE 17 Morphogen-Induced Synaptic Formation

As described in Examples 15 and 16, OP-1 induces dendritic growth invarious populations of cultured neurons. To determine if these dendritesare receptive to innervation, OP-1-treated cultured bippocampal neuronswere immunostained with antibodies to MAP2 and synapsin. Sites ofpresynaptic contact were defined by puncta of synapsin immunoreactivity.Given the poor growth of axons in cultured hippocampal neuronsmaintained in a serum-free medium, a heterochronic culture technique wasused to assess the ability of the OP-1-extended dendrites to receiveaxonal contacts. Cultured neurons were grown in the presence of OP-1 forthree days. New neurons were plated on top of these more mature neuronsand fixed one day later. Previous work has shown that axonal contactswill form within 24 hours of plating if more mature dendrites arepresent within the culture. Fletcher, et al., J. Neurosci 14: 6695-6706(1994). Using this heterochronic culture technique, synapsin positiveaggregates were found surrounding OP-1-induced dendrites. As illustratedin FIG. 12, OP-1-treated cultured hippocampal neurons had asignificantly higher number of synapses per neuron than untreatedneurons or neurons co-cultured with glial cells. These observationssuggest that the OP-1 induced dendritic outgrowth produces dendritesthat are receptive to innervation.

EXAMPLE 18 Morphogen-Induced Dendritic Growth and Synaptogenesis In Vivo

In order to assess the effects of morphogen on dendritic growth in vivo,rats are injected intraperitoneally once per day with OP-1 at dose of 2mg/kg. The control group consists of rats injected intraperitoneallywith the vehicle (20 mM arginine (pH 9.0), 150 mM NaCl with 0.1% Tween80). After seven days, rats are anesthetized with ether and the superiorcervical ganglia, hippocampus, and hypoglossal nucleus are removed.Subsequently, rats are perfused with paraformaldehyde and the kidney andretina are removed.

Superior cervical ganglia are desheathed and pinned in a chambersuperfused with an oxygenated physiological saline. For intracellularstaining, neurons are impaled with triangular glass electrodes filledwith a 4% solution of horseradish peroxidase (HRP). HRP is introducedinto the cell by iontophoresis and the reaction product is visualized bythe pyrocathecol-phenylenediamine method. Hanker, et al., Histochem. J9: 789-792 (1977); for details see Purves and Hume, J. Neurosci. 1:441-452 (1981); Forehand and Purves, J. Neurosci. 4: 1-12 (1984). Fiveto ten cells/ganglion are injected. After allowing two hours for dyediffusion, the ganglia are fixed in 4% formaldehyde overnight. Afterdehydration, stained neurons are viewed at 300×in whole-mountpreparations and traced with the aid of a camera lucida.

To confirm the light microscopic identification of processes and toassess the state of differentiation of the dendrites formed in thepresence of OP-1, superior cervical ganglia are immunostained withantibodies previously shown to react selectively with either axons ordendrites. Lein and Higgins, Dev. Biol. 136: 330-345 (1989). Monoclonalantibodies (mAb) to MAP2 (e.g., AP14), to nonphosphorylated forms of theM and H neurofilaments (SMI 32, Stembery-Meyer Immunocytochemicals), andto the transferrin receptor (MRC OX-26, Serotech) are used as dendriticmarkers and mAb to synaptophysin (SY-38, Boehringer Mannheim), tau(e.g., Tau 1), and phosphorylated forms of the H (NE14, BoehringerMannheim) and the M and H (SMI 31, Sternbery-Meyer Immunocytochemicals)neurofilament subunits are used as axonal markers. All antigens arelocalized by indirect immunofluorescence using previously describedprocedures. Lein and Higgins, Dev. Biol. 136: 330-345 (1989). Image 1Software (Universal Imaging) is used for the morphometric analyses ofdendritic growth in immunostained cultures. In addition, in order todetermine the effects of OP-1 on synaptogenesis in superior cervicalganglia in vivo, neurons are immunostained with antibodies to synapsin.Sites of presynaptic contact are defined by puncta of synapsinimmunoreactivity.

Hippocampal or hypoglossal tissue is impregnated with GolgiCox solution.Following dehydration, the tissue is embedded in celloidin and sectionedat 160 lm on a microtome. Sections are then developed in 5% sodiumsulphite and mounted on a glass slide with permount. Kidney and retinaltissue is removed from animals that have been perfused withformaldehyde. The fixed tissue is embedded in paraffin and sectioned at160 μm on a microtome. Sections are then developed in 5% sodium sulphiteand mounted on a glass slide with permount. Sections of hippocampal,hypoglossal, kidney, and retinal tissue are immunostained withantibodies previously shown to react selectively with axons, dendrites,or synapsin. Antigens are localized by indirect immunofluorescence, asdescribed above.

Dendritic and axonal processes are distinguished using establishedcriteria. Purves and Hume, J. Neurosci. 1: 441-452 (1981). Dendriteshave numerous short processes arising from the main shaft and branchedinto secondary and tertiary segments relatively close to the cell soma.The axon is readily identified as a smooth, thick process that usuallycould be followed for at least several hundred microns and frequentlycan be seen exiting the ganglion via a postganglionic nerve. The arborof each neuron is assessed by four measures of dendritic complexity. Thenumber of primary dendrites is determined by viewing the cells at 480×inmultiple focal planes. A primary dendrite is defined as any processextending from the soma a distance greater than one cell diameter. Totaldendritic lengths are measured from the camera lucida tracings with theaid of a digitizing tablet and a general purpose program for neuralimaging. Voyvodic, Soc. Neurosci. Abstr. 12: 390 (1986). The radius of acircle incorporating the entire arbor is measured as an indicator of theprocess length. Finally, the extent of branching is determined bycounting the number of branches crossing a 50% circle. Scholl, J. Comp.Neurol. 244: 245-253 (1953). Sites of presynaptic contact are defined bypuncta of synapsin immunoreactivity.

Animals treated with OP-1 are expected to have significantly enhanceddendritic growth when compared to control animals, reflected inincreased length, diameter, and number of processes. Further, animalstreated with OP-1 are expected to have significantly increased number ofsynaptic contact when compared to control animals.

EXAMPLE 19 Intra-Ocular Transplants

Intra-ocular grafting is a well established model which offers anisolated environment in which CNS synaptic contact can be selectivelyactivated and pharmacologically characterized using drug superfisiontechniques in vivo. See, for example, Olson, et al., ADVANCES INCELLULAR NEUROBIOLOGY, (Academic Press, 1983) Grafts of identified CNSareas are placed into the anterior eye chamber of syngeneic andallogeneic host rats. The development and overall structuralorganization of the graft is relatively organotypic in nature and themature transplant usually provides an in vivo replica of the gratedarea. Hoffer, et al., Brain Res. 79:165-184 (1974). After maturation ofthe transplant, host animals can be anesthetized and pre andpostsynaptic activity can be examined using in vivo electrochemical andelectrophysiological techniques. Eriksdotter-Nilsson et al., Brain Res.478: 269-280 (1989); Eriksdotter-Nilsson, et al., Exp. Brain Res. 74:89-98 (1989); Hoffer, et al., Brain Res. 79: 165-184 (1974); Johansson,et al., Exp. Neurol. 134: 25-34 (1995). After sacrifice of the hostanimal, grafts and underlying irides can be processed for histochemicalevaluations of neural and glial elements, and for localization ofvarious transmitter-specific structures and receptors. Björklund, etal., Dev. Brain Res. 6: 130-140 (1983); Bergman, et al., Hippocampus 2:339-348 (1992); Henschen, et al., Prog. Brain Res. 78: 187-191 (1988);Henschen, et al., Neuroscience 26: 193-213 (1988); and Henschen, et al.,Brain Res. 36: 237-247 (1988). Using sequential grafting of fetal braintissue pieces to the anterior chamber of the eye, it is possible tostudy the conditions under which mature brain and spinal cord tissue(grafts which have resided in oculo for one or more months and which nolonger show morphological signs of growth or development) will acceptingrowth of nerve fibers. Olson, et al., Brain Res. Bull. 9: 519-537(1982). Thus, using the in oculo technique, isolated replicas of definedpathways suitable for structural and functional studies of CNSconnectivity can be obtained.

In particular, it has been previously shown that grafted spinal cordwill survive and grow in oculo in a manner suggesting that is possessesa considerable intrinsic determination of its normal development.Henschen, et al., Exp. Brain Res. 60: 38-47(1985). Further, it has beenpreviously demonstrated that models of the descending coeruleo-spinalnoradrenergic and bulbospinal serotonergic pathways to spinal cord canbe generated when these CNS areas are co-grafted in oculo. Henschen, etal., Brain Res. Bull. 15: 335-342 (1985); Henschen, et al., Brain Res.Bull 17: 801-808 (1986); Henschen, et al., Dev. Brain Res. 36: 237-247(1987); Henschen, et al., Exp. Brain Res. 75: 317-326 (1989). In asimilar vein, corticospinal and sensory and motor pathways can beconstructed in oculo, using co-grafts of cerebral cortex (Palmer, etal., Exp. Brain Res. 87: 96-107 (1991); dorsal root ganglia (Trok, etal., 1997), and muscle (Trok, et al., Brain Res. 659: 138-146 (1994),with subsequent histological and electrophysiological analysis. Thus, inoculo grafts provide a unique isolated system of a much more “in vivo”than “in vitro” nature, to study spinal cord connectivity and responseto injury.

In oculo transplants of specific CNS areas have been employed toevaluate of various putative neurotrophic effects: NGF in hippocampus(Eriksdotter-Nilsson et al., Brain Res. 478: 269-280 (1989) andEriksdotter-Nilsson, et al., Exp. Brain Res. 74: 89-98 (1989)), FGF incortical areas (Giacobini, et al., Exp. Brain Res. 86: 73-81 (1991)),IGF-1 in olfactory bulb (Giacobini, et al., 1995)), GDNF in midbraindopaminergic nucleus and spinal cord (Johansson, et al., Exp. Neurol.134: 25-34 (1995); Trok, et al., Neuroscience 71: 231-241 (1996); andTrok, et al., (1996)), and BDNF, NT-3, NT-4, and CNTF in spinal cord(Trok, et al., 1997).

20.1 Effects of OP-1 on Intra-Ocular Spinal Cord Transplants

In oculo transplants Were employed to evaluate the effects of morphogenson motor neurons. Fisher 344 rats were implanted with syngeneic E18spinal cord grafts, essentially as previously described by Henschen, etal., Prog Brain Res. 78: 187-191 (1988). OP-1 (0.5 μg) or vehicle wasinjected into the anterior chamber at weekly intervals. Each rat had anOP-1-treated graft in one eye and a control graft in the contralateraleye. Survival and growth of the graft was followed noninvasively byobservation through the cornea over a four week period. After sacrificeof the host animals, grafts were evaluated by histological andimmunocytochemical techniques. Olson, L., et al., ADVANCES IN CELLULARNEUROBIOLOGY, pp. 407-442 (Academic Press, 1983); Granholm, et al., Exp.Neurol. 118: 7-17 (1992).

As shown in FIG. 13, OP-1-treated grafts maintained a significantlylarger size over the four week observation period, compared to controlgrafts. Transplants treated with weekly injections of vehicle hadminimal survival but manifested a marked reduction in size, similar towhat has been previously described for E18 donors. Henschen, et al.,Prog. Brain Res. 78: 187-191 (1988); Henschen, et al., Neuroscience 26:193-213 (1988). In contrast, grafts treated with weekly injections of0.5 μg of OP-1 maintained a much greater size and, at the end of fourweeks, had a size equal to, or only slightly less than, the initial sizeat grafting.

This positive effect of OP-1 was confirmed using immunocytochemicaltechniques. Overall neuron density was evaluated using MAP2 (i.e.,neurofilament immunoreactivity). As seen in FIG. 14, the number ofneurofilament-positive neuronal structures was significantly higher inOP-1-treated grafts compared to vehicle-treated transplants. In order toassess more specifically the effects of OP-1 on motor neurons,immunocytochemical studies were carried out using cholineacetyltransferase (CHAT) immunocytochemistry. The number of cholinergiccell bodies and fibers was also significantly higher in OP-1-treatedgrafts than in vehicle-treated transplants. See FIG. 15.

EXAMPLE 20 Traumatic Injury Model

The fluid percussion brain injury model was used to assess the abilityof morphogens to restore central nervous system functions followingsignificant traumatic brain injury.

I. Fluid Percussion Brain Injury Procedure

The animals used in this study were male Sprague-Dawley rats weighing250-300 grams (Charles River). The basic surgical preparation for thefluid-percussion brain injury has been previously described. Dietrich,et al., Acta Neuropathol. 87: 250-258 (1994) incorporated by referenceherein. Briefly, rats were anesthetized with 3% halothane, 30% oxygen,and a balance of nitrous oxide. Tracheal intubation was performed andrats were placed in a stereotaxic frame. A 4.8-mm craniotomy was thenmade overlying the right parietal cortex, 3.8 mm posterior to bregma and2.5 mm lateral to the midline. An injury tube was placed over theexposed dura and bonded by adhesive. Dental acrylic was then pouredaround the injury tube and the injury tube was then plugged with agelfoam sponge. The scalp was sutured closed and the animal returned toits home case and allowed to recover overnight.

On the next day, fluid-percussion brain injury was produced essentiallyas described by Dixon, et al., J. Neurosurg 67: 110-119 (1987) andClifton, et al., J Cereb. Blood Flow Metab. 11: 114-121 (1991). Thefluid percussion device consisted of a saline-filled Plexiglas cylinderthat is fitted with a transducer housing and injury screw adapted forthe rat's skull. The metal screw was firmly connected to the plasticinjury tube of the intubated anesthetized rat (70% nitrous oxide, 1.5%halothane, and 30% oxygen), and the injury was induced by the descent ofa pendulum that strikes the piston. Rats underwent mild-to-moderate headinjury, ranging from 1.6 to 1.9 atm. Brain temperature was indirectlymonitored with a thermistor probe inserted into the right temporalismuscle and maintained at 37-37.5° C. Rectal temperature was alsomeasured and maintained at 37° C. prior to and throughout the monitoringperiod.

II. Administration of Morphogen

Animals in the treatment group received OP-1 intracisternally at a doseof 10 μg/injection. Control animals received vehicle solutions lackingOP-1 but with all other components at equivalent final concentrations.Both OP-1 and vehicle-treated animals received two injections, one dayand four days following the fluid percussion injury.

To administer the injection, the animals were anesthetized withhalothane in 70% NO₂/30% O₂ and placed in a stereotaxic frame. Theprocedure for intracisternal injection of OP-1 containing solutions orvehicle-only solutions was identical. Using aseptic technique, OP-1(1 or10 μg/injection) or an equivalent volume of vehicle were introduced bypercutaneous injection (10 μl/injection) into the cisterna magna using aHamilton syringe fitted with a 26 gauge needle (Yamada, et al., (1991)J. Cereb. Blood Flow Metab. 11: 472-478). Before each injection, 1-2 μlof cerebrospinal fluid (CSF) was drawn back through the Hamilton syringeto verify needle placement in the subarachnoid space. Preliminarystudies demonstrated that a dye, 1% Evans blue, delivered in thisfashion diffused freely through the basal cisterns and over the cerebralcortex within one hour of injection. Animals were randomly assigned toeither of the OP-1 treatment groups or to the vehicle treatment group.Animals received two intracistemal injections (2×10 μg/injection OP-1 or2×vehicle); the first injection was administered 24 hours after thebrain injury and the second injection was administered 4 days after thebrain injury.

III. Behavioral Testing

Three standard functional/behavioral tests were used to assesssensorimotor and reflex function after brain injury. The tests have beenfully described in the literature, including Bederson, et al., (1986)Stroke 17: 472476; DeRyck, et al., (1992) Brain Res. 573: 44-60;Markgraf, et al., (1992) Brain Res. 575: 238-246; and Alexis, et al.,(1995) Stroke 26: 2338-2346.

A. The Forelimb Placing Test

Forelimb placing to three separate stimuli (visual, tactile, andproprioceptive) was measured to assess sensorimotor integration. DeRyck,et al., Brain Res. 573:44-60 (1992). For the visual placing subtest, theanimal is held upright by the researcher and brought close to a tabletop. Normal placing of the limb on the table is scored as “0,” delayedplacing (<2 sec) is scored as “1,” and no or very delayed placing (>2sec) is scored as “2.” Separate scores are obtained first as the animalis brought forward and then again as the animal is brought sideways tothe table (maximum score per limb=4; in each case higher numbers denotegreater deficits). For the tactile placing subtest, the animal is heldso that it cannot see or touch the table top with its whiskers. Thedorsal forepaw is touched lightly to the table top as the animal isfirst brought forward and then brought sideways to the table. Placingeach time is scored as above (maximum score per limb=4). For theproprioceptive placing subtest, the animal is brought forward only andgreater pressure is applied to the dorsal forepaw; placing is scored asabove (maximum score per limb=2). Finally, the ability of animals toplace the forelimb in response to whisker stimulation by the tabletopwas tested (maximum score per limb 2). Then subscores were added to givethe total forelimb placing score per limb (range=0-12).

B. The Beam Balance Test

Beam balance is sensitive to motor cortical insults. This task was usedto assess gross vestibulomotor function by requiring a rat to balancesteadily on a narrow beam. Feeney, et al., Science, 217: 855-857 (1982);Goldstein, et al., Behav. Neurosci. 104: 318-325 (1990). The testinvolved three 60-second training trials 24 hours before surgery toacquire baseline data. The apparatus consisted of a ¾-inch-wide beam, 10inches in length, suspended 1 ft. above a table top. The rat waspositioned on the beam and had to maintain steady posture with all limbson top of the beam for 60 seconds. The animals' performance was ratedwith the scale of Clifton, et al., J. Cereb Blood Flow Metab. 11:114-121(1991), which ranges from 1 to 6, with a score of 1 being normaland a score of 6 indicating that the animal was unable to support itselfon the beam.

C. The Beam Walking Test

This was a test of sensorimotor integration specifically examininghindlimb function. The testing apparatus and rating procedures wereadapted from Feeney, et al., Science, 217: 855-857 (1982). A 1-inch-widebeam, 4 ft. in length, was suspended 3 ft. above the floor in a dimlylit room. At the far end of the beam was a darkened goal box with anarrow entryway. At equal distances along the beam, four 3-inch metalscrews were positioned, angling away from the beam's center. A whitenoise generator and bright light source at the start of the beammotivated the animal to traverse the beam and enter the goal box. Onceinside the goal box, the stimuli were terminated. The rat's latency toreach the goal box (in seconds) and hindlimb performance as it traversedthe beam (based on a 1 to 7 rating scale) were recorded. A score of 7indicates normal beam walking with less than 2 foot slips, and a scoreof 1 indicates that the rat was unable to traverse the beam in less than80 seconds. Each rat was trained for three days before surgery toacquire the task and to achieve normal performance (a score of 7) onthree consecutive trials. Three baseline trials were collected 24 hoursbefore surgery, and three testing trials were recorded daily thereafter.Mean values of latency and score for each day were computed.

IV. Results

As illustrated in FIGS. 16-18, OP-1 enhanced the recovery from traumaticbrain injury in all three behavioral measures. In the forelimb placingtests, OP-1-treated animals showed a gradual decrease in injury severityscores which attained statistical significance by day 9. See FIG. 16. Inthe beam balance and beam walking tests, OP-1-treated animals hadperformance scores that were essentially identical to sham-controlanimals. See FIGS. 17 and 18. These observations suggests thatmorphogens are capable of restoring impaired or lost sensory-motorfunctions following a traumatic brain injury, including visual, tactile,and proprioceptive placement, gross vestibulomotor function, andsensorimotor integration.

Similar routine modifications can be made in other accepted models oftraumatic central nervous system injury, to confirm efficacy ofmorphogen treatment to restore impaired or lost sensory-motor functions.

1-9. (Canceled)
 10. A method of preserving motor function in a mammalwith symptoms of or at risk of amyotrophic lateral sclerosis, comprisingadministering to said mammal a morphogen, wherein the morphogen: (1)comprises a dimeric protein having an amino acid sequence with: (a) atleast 70% homology with the C-terminal seven-cysteine skeleton of humanOP-1, residues 330-431 of SEQ ID NO: 2; (b) having greater than 60%amino acid sequence identity with said C-terminal seven-cysteineskeleton of human OP-1; (c) defined by Generic Sequence 7, SEQ ID NO: 4;(d) defined by Generic Sequence 8, SEQ ID NO: 5; (e) defined by GenericSequence 9, SEQ ID NO: 6; (f) defined by Generic Sequence 10, SEQ ID NO:7; or (g) defined by OPX, SEQ ID NO: 3; and (2) stimulates production ofan N-CAM or L1 isoform by an NG108-15 cell in vitro; whereby motorfunction is preserved in said mammal.
 11. (Canceled)
 12. A method ofpreserving motor function in a mammal with symptoms of or at risk of aspinal cord injury, comprising administering to said mammal a morphogen,wherein the morphogen: (1) comprises a dimeric protein having an aminoacid sequence with: (a) at least 70% homology with the C-terminalseven-cysteine skeleton of human OP-1, residues 330-431 of SEQ ID NO: 2;(b) greater than 60% amino acid sequence identity with said C-terminalseven-cysteine skeleton of human OP-1; (c) defined by Generic Sequence7, SEQ ID NO: 4; (d) defined by Generic Sequence 8, SEQ ID NO: 5; (e)defined by Generic Sequence 9, SEQ ID NO: 6; (f) defined by GenericSequence 10, SEQ ID NO: 7; or (g) defined by OPX, SEQ ID NO: 3; and (2)stimulates production of an N-CAM or L1 isoform by an NG108-15 cell invitro; whereby motor function is preserved in said mammal. 13-18.(Canceled)
 19. A method of preserving motor function in a mammal withsymptoms of or at risk of amyotrophic lateral sclerosis, comprisingadministering to said mammal a morphogen selected from: human OP-1,mouse OP-1, human OP-2, mouse OP-2,60A, GDF-1, BMP2A, BMP2B, DPP, Vgl,Vgr-1, BMP3, BMP5, or BMP6, wherein said morphogen stimulates productionof an N-CAM or L1 isoform by an NG108-15 cell in vitro whereby motorfunction is preserved in said mammal.
 20. (Canceled)
 21. A method ofpreserving motor function in a mammal with symptoms of or at risk of aspinal cord injury, comprising administering a morphogen selected from:human OP-1, mouse OP-1, human OP-2, mouse OP-2, 60A, GDF-1, BMP2A,BMP2B, DPP, Vgl, Vgr-1, BMP3, BMP5, or BMP6, wherein said morphogenstimulates production of an N-CAM or L1 isoform by an NG108-15 cell invitro whereby motor function is preserved in said mammal. 22-23.(Canceled)
 24. The method of claim 10, wherein the morphogen comprises adimeric protein having an amino acid sequence with at least 70% homologywith the C-terminal seven-cysteine skeleton of human OP-1, residues330-431 of SEQ ID NO:
 2. 25. The method of claim 10, wherein themorphogen comprises a dimeric protein having an amino acid sequence withgreater than 60% amino acid sequence identity with said C-terminalseven-cysteine skeleton of human OP-1.
 26. The method of claim 12,wherein the morphogen comprises a dimeric protein having an amino acidsequence with at least 70% homology with the C-terminal seven-cysteineskeleton of human OP-1, residues 330-431 of SEQ ID NO:
 2. 27. The methodof claim 12, wherein the morphogen comprises a dimeric protein having anamino acid sequence with greater than 60% amino acid sequence identitywith said C-terminal seven-cysteine skeleton of human OP-1.